L-Threonine derivatives of high therapeutic index

ABSTRACT

The present invention is directed to a derivative comprised of an L-Threonine bonded to a medicament or drug having a hydroxy, amino, carboxy or acylating derivative thereon. The derivative has the same utility as the drug from which it is made, but it has enhanced therapeutic properties. In fact, the derivatives of the present invention enhance at least one or more therapeutic qualities, as defined herein. The present invention is also directed to pharmaceutical compositions containing same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U.S. patentapplication Ser. No. 11/343,557, filed Jan. 30, 2006, which is acontinuation-in-part of PCT Application No. PCT/US04/24901, which claimsbenefit of Provisional Application No. 60/491,331, filed Jul. 29, 2003.This application incorporates by reference the subject matter disclosedin both U.S. Ser. No. 11/343,557 and PCT/US04/24901 in their entirety.

FIELD OF THE INVENTION

The invention relates to chiral separation of racemic and diastereomericpharmaceutical compounds using L-Threonine, a naturally occurring aminoacid and to methods of treating particular ailments, which areameliorated by the administration of these enantiomerically anddiastereomerically pure drugs and/or their corresponding L-Threonineester/amide derivatives and to pharmaceutical compositions containingthese substantially pure drugs and substantially pure L-Threoninederivatives.

The current invention involves improving many physicochemical,biopharmaceutical, and clinical efficacy of various drugs usingL-Threonine as covalently bonded carriers for these drugs, with theadditional advantage of separating various enantiomeric anddiastereomeric drugs into their constitutent individual isomers.

BACKGROUND OF THE INVENTION

Chirality in organic and pharmaceutical chemistry plays a major role.While a vast majority of the new drugs introduced in the globalpharmaceutical arena are chiral drugs and are resolved, there are anumber of drugs in various therapeutic category that are still racemicand diastereromeric mixtures, such as Non-Steroidal Anti-Inflammatorydrugs (NSAIDs) based on the structure of aryl propionic acid such asibuprofen and other classes of drugs such as labatelol.

Drugs work in the mammalian body with so called pharmacological“receptors” that have specific shape whereby the drug molecules can onlyfit into these receptors like a “glove”. Since it is not possible tosuperimpose a left handed glove on a right handed glove, the mirrorimages of the molecules are not superimposable.

The development of chemical compounds for the treatment of disorders,maladies and diseases has become increasingly difficult and costly. Theprobability of success for such development is often discouragingly low.Further, the time for development can approach or exceed ten years,leaving large numbers of patients without remedy for an extended periodof time. In addition, the costs of developing a new drug for thetreatment of any malady of significance might exceed a Billion dollarsin a few years.

Even in cases in which effective pharmaceutical compounds have beendeveloped, there are often disadvantages associated with theiradministration. These disadvantages can include aesthetic,biopharmaceutic, and pharmacokinetic bafflers affecting theeffectiveness of some existing pharmaceutical compounds. For example,unpleasant taste or smell of a pharmaceutical compound or compositioncan be a significant barrier to patient compliance with respect to theadministration regimen. The undesirable solubility characteristics of apharmaceutical compound can also cause difficulty in the formulation ofa homogeneous composition. Other disadvantages associated with knownpharmaceutical compounds include: poor absorption of orally administeredformulations; poor bioavailability of the pharmaceutical compounds inoral formulations; lack of dose proportionality; low stability ofpharmaceutical compounds in vitro and in vivo; poor penetration of theblood/brain barrier; excessive first-pass metabolism of pharmaceuticalcompounds as they pass through the liver; excessive enterohepaticrecirculation; low absorption rates; ineffective compound release at thesite of action; excessive irritation, for example, gastro-intestinalirritability and/or ulceration; painful injection of parenterallyadministered pharmaceutical compounds and compositions; excessively highdosages required for some pharmaceutical compounds and compositions, andother undesirable characteristics. Some pharmaceutical compounds areprocessed by the body to produce toxic by-products with harmful effects.

The art is continually seeking new chemical compounds for the treatmentof a wide variety of disorders, with improved properties to overcome thedisadvantages of known pharmaceutical compounds mentioned above.

The present invention has overcome many problems associated withcurrently marketed drugs by making a derivative thereof. The concept ofderivatives is well known, and there are a number of examples of suchderivatives enumerated in the literature and there are a number ofderivatives available in the market, including such diverse groups asstatin drugs, ACE inhibitors, antiviral drugs such as Acyclovir and thelike.

The present invention, however, uses specifically L-Threonine as themoiety to make the derivatives.

SUMMARY OF THE INVENTION

The present invention is directed to pharmaceutically active pureenantiomers of a derivative, having L-Threonine covalently bonded to apharmaceutical compound (drugs) to form said acid derivative, which isadministered in this form to the subject, such as a mammal. It is alsodirected to pharmaceutical compositions comprising a therapeuticallyeffective amount of a L-Threonine covalently bounded to a drug and apharmaceutical carrier therefor. It is also directed to the method ofuse of such drugs thus formed. The utility of the drug to which theL-Threonine moiety is attached is the same as that of the underlyingdrug from which the L-Threonine derivative is prepared.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically compares the efficacy of L-serine ester of (±)Ibuprofen (F1), L-threonine ester (±) Ibuprofen (F2) andL-hydroxyproline ester of (±) Ibuprofen (F3), (±) Ibuprofen (i.e., theracemic mixture) and Ibuprofen (S)(+), after one hour dosing, based onthe antagonizing property of Acetylcholine induced writhe in Albinomice.

FIG. 2 graphically compares the efficacy of L-serine ester of (±)Ibuprofen, (F1), L-Threonine ester of, (I) Ibuprofen (F2),L-hydroxyproline ester of (±) Ibuprofen (F3), ± Ibuprofen and S(+)Ibuprofen after 3 hour dosing, based on the antagonizing property ofAcetylcholine induced writhes in albino mice.

FIGS. 3-8 compares graphically the plasma concentration in humans afteradministration of ibuprofen and ibuprofen Threonine ester.

FIG. 9 depicts graphically the mean clotting time of acetylsalicyclicacid and L-serine, L-Threonine and L-hydroxy proline acetylsalicyclicacid. In FIG. 9, F1, F2 and F3 are respectively, ASA-Serine,ASA-Hydroxyproline and ASA-Threonine esters.

FIG. 10 shows clotting time in minutes versus doses administered to ratsat 1,4 and 10 mg/kg for test drugs, ASA-Serine Ester, ASA-HydroxyprolineEster and ASA-Threonine Ester versus Aspirin as the reference standard.

FIG. 11 depicts the average Clotting time after 325 mg and 81 mgAcetylsalicylic Acid-L-Threonine Ester and Bayer Aspirin is administeredto human Volunteers. The first set of columns (Normal) is the averageclotting times observed prior to administration of the Aspirinderivative and Aspirin.

FIG. 12 shows clotting time in minutes—5 day administration of 81 mgACETYLSALICYLIC ACID-L-THREONINE ESTER and Bayer Aspirin in HumanVolunteers. Total of 3 volunteers participated in this study. Twovolunteers took ACETYLSALICYLIC ACID-L-THREONINE ESTER (bottom line inFIG. 12).

FIG. 13 graphically depicts percentage increase in clotting time ofAcetylsalicylic Acid-L-Threonine Ester vs. Aspirin (Bayer) at 81 mg dosebased on 5-day average increase. The two AcetylsalicylicAcid-L-Threonine Ester blocks shown correspond to two separatevolunteers who took the test drug over a period of 5 days. The thirdvolunteer took Bayer Aspirin for 5 days.

FIG. 14 is a plot of the concentration of Aspirin versus time in 4volunteers who took Acetylsalicylic Acid-L-Threonine Ester and 2volunteers who took Bayer Aspirin.

FIG. 15 is a plot of the plasma concentration of Salicylic Acid versustime in human plasma for 4 volunteers who took AcetylsalicylicAcid-L-Threonine Ester and two volunteers who took Aspirin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In one embodiment, the present invention is directed to L-threoninederivatives of drugs. L-Threonine is an ideal model to be used as aderivative, because it is capable of forming various types of linkagesbetween itself and the drug. By definition, an L-Threonine has at leasttwo functionalities thereon, an amino group (NH₂) and a carboxy group(COOH). For example, the α-L-Threonines have the well known structure

wherever R₀ is the side group or chain of the L-Threonine, i.e.,CH(OH)(CH₃). The

as defined herein, is the main chain of the L-Threonine. Thus, forexample, besides the amino group and the carboxyl group on the mainchain, the side chain has a functional group thereon, an OH group. It isthe functional groups on the L-Threonine moiety that permits thecovalent linkage to occur between the L-Threonine and the drug.

The drug or medicament useful in the present invention containsfunctional groups thereon that permit the drug to react with and form acovalent bond with the L-Threonine. Examples of functional groupspresent on the drugs include NH₂, OH, COOH or acid derivatives thereof,such as esters, amides and the like.

The mode of attachment between the pharmaceutical compound and theL-Threonine can be via:

1) An ester bond (—CO—O—) arising from condensation of a carboxylic acidand an alcohol or phenolic hydroxyl group, or throughtransesterification, for example:

-   -   a) Where the pharmaceutical compound has an aliphatic or        aromatic hydroxyl group an ester bond can be formed with the        backbone carboxylic acid group of L-Threonine under        esterification conditions; or    -   b) Where the pharmaceutical compound has an aliphatic or        aromatic hydroxyl group and the L-Threonine has the alcohol        functionality on the side chain oxidized to an carboxylic acid,        an ester bond can be formed therebetween under esterification        conditions; or    -   c) Where the pharmaceutical compound has a carboxylic acid group        and the L-Threonine has a side chain aliphatic or aromatic        hydroxyl group, an ester bond can be formed therebetween under        esterification condition; or    -   d) Where the pharmaceutical compound has an ester group with a        substituted or unsubstituted acyloxy (e.g., alkoxy- or        arylalkoxy-, aryloxy carbonyl) substituent        (compound-O—CO-substituent) and the L-Threonine has a backbone        carboxylic acid group, an ester bond can be formed therebetween        through transesterification; or    -   e) Where the pharmaceutical compound has an ester group with a        substituted or unsubstituted acyloxy (e.g., alkoxy- or        arylalkoxy-, aryloxy carbonyl) substituent        (compound-O—CO-substituent) and the L-Threonine side chain has        been oxidized to a carboxylic acid group, an ester bond can be        formed therebetween through transesterification; or    -   f) Where the pharmaceutical compound has an ester group with a        substituted or unsubstituted alkoxy- or arylalkoxy- or aryloxy        carbonyl substituent (compound-CO—O-Substituent) and the        L-Threonine has a side chain hydroxyl group, an ester bond can        form therebetween though transesterification; or

2) An amide bond (—CO—NH—) arising from condensation of a carboxylicacid and an amine, for example:

-   -   a) Where the pharmaceutical compound has an amino group and the        L-Threonine has a backbone carboxylic acid group, an amide can        be formed under amide forming conditions; or    -   b) Where the pharmaceutical compound has an amino group and the        L-Threonine side chain has been oxidized to a carboxylic acid        group, an amide bond can form therebetween under amide forming        conditions; or    -   c) Where the pharmaceutical compound has a carboxylic acid group        and the L-Threonine has a backbone amino group, an amide bond        can form therebetween under amide forming conditions; or    -   d) Where the pharmaceutical compound has a carboxylic acid group        and the L-Threonine hyroxy group on the side chain has been        converted to an amino group, for example, by nucleophilic        substitution and, an amide bond can be formed therebetween under        amide forming conditions.

Thus, the present invention is directed to the derivatives thus formed.A novel result of the present invention is that the naturally occurringL-Threonine was used to form the derivatives of a various classes ofdrugs containing —COOH, —NH or —OH groups.

The derivatives of the present invention have a number of advantages.For example, when L-Threonine derivatives are administered by a numberof routes such as oral, IV, rectal or other such methods, thesederivatives are converted into active drug molecules. A significantadvantage of the L-Threonine derivative is that it is non-toxic, andhence either assimilated into the body or safely excreted. This is quiteunlike the characteristics exhibited by a number of derivativesavailable in the market, where the promoiety itself is toxic, as is thecase with statin drugs, Enalapril, Benazapril and the like group of aceinhibitors, and a number of antibiotics such as pivoxil, Axetil,Cilexetil and the like groups, which are highly toxic, thereby reducingthe therapeutic index of the active drug. Moreover, the L-Threoninederivatives of the present invention are significant in the fact thatthey can separate the racemic mixtures of a number of drugs. It can dothis separation at minimal cost, and also it is very surprising to notethat only L-Threonine is able to separate racemic mixtures of variousdrugs, and other hydroxyl group containing naturally occurring hydroxycontaining amino acids, such as L-Serine or L-hydroxyproline do not havethe same capability as described below. More specifically, it has beenfound that such threonine derivatives are easily separated as pureisomers from the required enantiomeric mixture. Furthermore, as shownhereinbelow the derivative thus formed has advantages not realizedrelative to the drug without the L-Threonine attached thereto. Forexample, it can improve bioavailability, efficacy, be less toxic,exhibit greater solubility in water and/or improve the ability of thedrug to pass into the cell membrane or through blood brain barrier,exhibit less side effects, such as gastro-intestinal irritability,enhanced therapeutic index and the like.

Thus, the present invention is directed to a method of improving thetherapeutic quality of a drug wherein the improvement in the therapeuticquality is selected from the group consisting of improved efficacy,enhanced therapeutic index, increased solubility in the mammal'sinternal fluid, improved passage through the cell membrane, improvedpassage through the blood brain barrier, decreased side effects, such assignificantly decreased irritation and/or ulcerations, less toxicity,enhanced absorption ratio and the like relative to the correspondingdrug administered to the patient in the non-derivative form, said methodcomprising reacting the drug with an L-Threonine to form a covalent bondtherebetween and administering the product thereof (hereinafter“derivative”) to a patient. The derivatives of the present inventionhave at least one of the aforementioned improved qualities. In fact,they exhibit preferably at least two of the improved qualities citedhereinabove. Other advantages of the L-Threonine derivatives of thedrugs of the present invention include the wide availability of theL-Threonines and the ease in which the reactions take place. Forexample, when L-threonine is reacted with the drug to form an amide orother ester, the reaction to form the amide or ester is generally moreefficient and yields are very high, presumably above about 70% and morepreferably above about 80% and most preferably above about 90%.

Most importantly, the preparation of the compounds of the presentinvention readily sepearates the enantiomers from a racemic mixture. Forinstance, it is well known in the art, when a drug is presented as anenantiomeric mixture, only one optically active form is responsible forthe drug's pharmacological activity. This phenomenon has been repeatedlydemonstrated for the whole class of NSAIDs, and ACE inhibitors, statindrugs and other therapeutic categories. The present inventionsurprisingly is able to separate the enantiomers without much additionalwork using L-Threonine, the naturally occurring amino aicd, and theresulting derivative also shows significant improvement in therapeuticefficacy.

Thus, L-Threonine is useful not only to separate the racemic mixtures ofpharmacologically active drugs to their corresponding individualisomers, but it also produces useful derivatives that arepharmacologically and biopharmaceutically superior to the correspondingparent active drugs.

As used here, the terms “drug”, “medicament”, and “pharmaceutical” arebeing used interchangeably and refer to the active compound that isadministered to the patient without attachment of the L-Threoninethereto. Moreover, as used herein, the drug contains a functional groupthereon capable of reacting with the L-Threonine, such as NH₂, OH, COOHor acylating derivatives thereof (e.g., ester, anhydride, amide, and thelike) and the like.

The drug may be a peptide or contain a peptide. In accordance with thepresent invention, the threonine does not replace one of the amino acidsin the peptide. Nor does the L-threonine derivative refer to the drugcomprised of a peptide containing a L-threonine moiety on the molecule.On the contrary, as described herein, just as with any other drug, theL-threonine moiety is added to the drug by covalently bonding it to thepeptide, either on a side chain or preferably with the amino group orN-amino end or with the carboxy broup on the carboxy end of the peptide.When the drug is linked to an L-Threonine, the term “L-Threoninederivative” or “derivative of the present invention”, or “compound ofthe present invention” or synonym thereto is utilized.

Among the L-Threonines useful in reacting with the drugs, it has beenfound that naturally occurring L-Threonine is the most useful amino acidto separate enantiomeric mixtures of active drugs. One who is normallytrained in the art of understanding the medicinal chemistry andpharmacological activity would think that if L-Threonine is able toseparate enantiomeric mixtures of the active drugs, then other Hydroxylgroup containing amino acids such as Serine, Hydroxyproline or Tyrosine,should also be able to separate the enantiomeric mixtures. However,surprisingly, it has been found that L-Threonine is the most effectiveseparating the enantiomeric mixtures and that the other amino acids,including the hydroxy containing amino acids, do not so readily.

Thus, the inventor has found that Threonine is extremely effective whenit comes to the separation of enantiomers and diastereoisomers. Theabsolute configuration of L-Threonine is shown below:

[R—(R*,S*)]-2-amino-3-hydroxybutanoic acid

The following reaction schemes depict the reactions discussedhereinabove with respect to the reaction of hydroxyl, carboxyl and aminecontaining drugs with L-Threonine.

Reaction Scheme A: Where the hydroxyl group of the drug is reacted withthe carboxyl group of L-Threonine to from the ester derivative

Reaction Scheme B: Where the carboxyl group of the drug is reacted withthe hydroxyl group of L-Threonine wherein the hydroxy group is on theside chain to form the ester derivative.

Reaction Scheme C: Where the amine group of the drug is reacted with thecarboxyl group of L-Threonine to from the amide derivative

Reaction Scheme D: Where the carboxyl group of the drug is reacted withthe carboxyl group of the L-Threonine to form the anhydride derivative.

Reaction Scheme E: Where the amine group of the drug is reacted with theamine group of the L-Threonine to form the azo derivative derivative.

Reaction Scheme F: Where the carboxyl group of the drug is reacted withamine group of the L-Threonine to form the amide derivative.

As used herein the term “L-Threonine” refers to an organic compoundhaving therein a carboxyl group (COOH) and an amino group (NH₂) or saltsthereof in the L-configuration. In solution, these two terminal groupsionize to form a double ionized, through overall neutral entityidentified as zwitterions. The ionic ends are stabilized in aqueoussolution by polar water molecules.

The term L-Threonine or “acylating derivative” thereof refers to theL-Threonine amino acid or an acylating derivative thereof, such ashalide (e.g., Br, Cl, I or F), ester (e.g., lower alkylester, arylester, aryl lower alkyl ester, cycloalkyl ester, cycloalkylloweralkylester heterocyclic ester or lower alkylheterocyclic ester or ananhydride, e.g., N-carboxyanhydride of Threonine.

When the side group of the Threonine, the hydroxy group become involvedin the acylating reaction described above, the bond thus formed may bedepicted as OAA₁ wherein AA₁ is L-Threonine residue without the hydroxygroup, i.e.,

Thus, AA₁ by this definition, refers to the L-Threonine without thehydroxy side group since it took part in the reaction in forming theester. Moreover, when an ester is formed between the hydroxy group ofthe L-Threonine and the OH group of the drug, the hydroxy group on thecarboxy group forms a byproduct with the hydrogen of the hydroxy group,thus, the resulting product does not have the OH group on the carboxygroup, but just the acyl moiety.

On the other hand, the amide bond may be depicted as C(═O)—NHAA whereinAA is

this means that the L-Threonine forms an amide bond between the carboxygroup on the drug and the amino group of the L-Threonine. However, aswritten, since the NH from the amide bond comes from the L-Threonine, AAis the L-Threonine without the amino group. Finally, the bond may formbetween the carboxy group of the L-Threonine and the amino group orhydroxy group of the drug. In such a case, the molecule is depicted as

wherein AA₂ is

There appears to be only one preferred L-Threonine, the naturallyoccurring L-Threonine, whose structure is shown above. Only drugs thatare tertiary amines can not participate in formation of amides.Secondary amines can be reacted to form amides. Primary amines can bereacted with L-Threonine to form either azo bond or an amide linkage.

The derivatives are prepared from a drug having a group thereon whichcan react with the L-Threonine.

The preferred drugs that are reacted with L-Threonines in accordancewith various schemes are shown in the table below. This is onlyrepresentative examples, and not inclusive of all the drugs. ReactionSchemes Drug A B C D E F Abacavir YES YES YES Acarbose YES AcebutololYES YES Adefovir YES Albuterol YES YES Amlodipine¹ YES Amphotericin BYES YES YES YES Amprenavir YES YES YES Atenolol YES YES YES AtorvastatinYES YES YES YES Atropine YES Baclofen YES YES YES YES YES BenazeprilatYES YES YES YES Betaxolol YES YES Bicalutamide YES YES Biotin YES YESYES YES Biperiden YES Bisoprolol YES YES Bitolterol YES YES BrinzolamideYES YES Bupivacaine YES Buprenorphine YES Bupropion YES Butorphanol YESCapacitabine YES Captopril YES YES YES YES Carbidopa YES YES YES YES YESYES Carnitine YES YES YES YES Carteolol YES YES Cefditoren YES YES YESYES YES Cerivastatin YES YES YES YES Chloramphenicol YES Cisapride YESYES Clopidogrel Acid YES YES YES Clorazepic Acid YES YES YES YESCycloserine YES YES Cytarabine YES YES YES Danazol YES DextroamphetamineYES YES Didanosine YES YES Digoxin YES Divalproex YES YES YES DocetaxelYES YES Dorzolamide YES YES Dyphylline YES Dysopyramide YES YESEfavirenz YES Enalaprilat YES YES YES YES Ephedrine YES YES EplerenoneYES YES YES Eprosartan YES YES YES Esmolol YES YES Estramustine YESEthambutol YES YES Ethchlorvynol YES Ethosuximide YES Ethotoin YESEtidocaine YES Etoposide YES Ezetimibe YES Fenofibrate YES YES YESFenoprofen YES YES YES Fexofenadine YES YES YES YES Finasteride YESFluoxetine YES Fluticasone YES Fluvastatin YES YES YES YES Folic AcidYES YES YES YES YES Fosinoprilat YES YES Frovatriptan YES YESFulvestrant YES Gabapentin YES YES YES YES YES Ganciclovir YESGlimepiride YES Goserelin YES Hydroxychloroquine YES Hydroxyzine YESHyoscyamine YES Ibuprofen YES YES YES Ibutilide YES YES Indapamide YESYES Indinavir YES YES Ipratropium YES Irinotecan YES Isosorbide YESIsradipine² YES Ketoprofen YES YES YES Ketorolac YES YES YES LabetalolYES YES Lamivudine YES YES YES Lamivudine YES YES YES Lansoprazole YESLatanoprost Acid YES YES YES YES Leuprolide YES Levobunolol YES YES YESLevodopa YES YES YES YES YES YES Levorphanol YES Liothyronine YES YESYES YES YES YES Lisinopril YES YES YES YES YES Lopinavir YES YESLorazepam YES Lovastatin YES YES YES YES Medroxyprogesterone YESMefloquine YES YES Megestrol YES Mephobarbital YES Mepivacaine YESMetaproterenol YES YES Metformin YES YES Methamphetamine YESMethohexital YES Methotrexate YES YES YES Methylphenidate YES YES YESYES Methylphenidate³ YES Methylprednisolone YES Metolazone YES YESMetoprolol YES YES Mexiletine YES YES Miglitol YES Miglitol YESMoexiprilat YES YES YES YES Mometasone YES Montelukast YES YES YES YESNadolol YES YES Nalbuphine YES Naproxen YES YES YES Naratriptan YES YESNateglinide YES YES YES YES Nelfinavir YES YES Niacin YES YES YESNicardipine⁴ YES Nimidipine⁵ YES Nisoldipine⁶ YES Norgestimate YESOctreotide YES YES Ofloxacin YES YES YES Olmesartan YES YES YESOmeprazole YES YES Paclitaxel YES YES Pantothenic Acid YES YES YES YESYES Paroxetine YES YES Paroxetine YES Pemoline YES YES Penbutolol YESYES Penicillamine YES YES YES YES YES Pentazocine YES Pentobarbital YESPerindoprilat YES YES YES YES Phenylephrine YES YES PhenylpropanolamineYES YES YES Pindolol YES YES Pioglitazone YES Pirbuterol YES PramipexoleYES YES Pravastatin YES YES YES YES Propafenone YES YES Propranolol YESYES Pseudoephedrine YES YES Quinacrine YES Quinaprilat YES YES YES YESQuinethazone YES YES Quinidine YES Quinine YES Ramiprilat YES YES YESYES Reboxetine YES Repaglinide YES YES YES YES Repaglinide YES YES YESYES YES Ribavirin YES YES YES Ritonavir YES YES Ropivacaine YESRosiglitazone YES Rosuvastatin YES YES YES YES Salmeterol YES YESSertraline YES Simavastatin YES YES YES YES Sirolimus YES Sotalol YESYES Sulfa Drugs YES YES Sulfasalazine YES Sumitriptan YES YES TacrolimusYES Tazorotene YES YES YES Telmesartan YES YES YES Tenofovir YESTerbutaline YES YES Thyroxine YES YES YES YES YES Tiagabine YES YES YESTimolol YES YES Tirofiban YES YES YES YES Tocainide YES YES Tramadol YESTrandolaprilat YES YES YES YES Tranylcypromine YES YES Treprostinil YESYES YES YES Triamcinolone YES Troglitazone YES YES Unoprostone YES YESYES Valsartan YES YES YES Venlafaxine YES Vidarabine YES YES YESWarfarin YES Zalcitabine YES YES YES Zidovudine YES YES Zolmitriptan YESYES¹In case of Amlodipine, one can replace 5-methyl ester moiety with3-L-Threonine with better therapeutic index. In case of intactAmlodipine molecule, biotransformation results in generation of methanoldue to solvolysis of 5-methyl ester, which is highly toxic, andreplacement of this with L-Threonine results in a product with much lesstoxicity. Same argument goes for rest of the products in this categorystated below:²For Isradipine, replace 5 methyl ester with 5-L-Threonine ester. Themethyl ester is the active, and apparently the carboxylic acidderivative is not active.³Replacing the methyl group with L-Threonine will still maintainactivity, but none of the toxicity of methylphenidate.⁴For Nicardipine, replace 5-methyl ester with 5-L-Threonine for bettertherapeutic index.⁵For Nimodipine, one could replace 5 (1-methyl)ethyl ester with5-L-Threonine for better therapeutic index.⁶In case of Nisoldipine, replace 5-methyl ester with 5-L-Threonine forbetter therapeutic index, and no loss of activity.

It is to be understood that the base structure of each of the drugsenumerated herein is incorporated by reference. Moreover, the productsthereof, although not drawn, are understood by one of ordinary skill inthe art, based upon the reaction schemes described hereinabove and areconsidered as part of this disclosure.

The derivatives of the present invention may be L-Threonineesters/amides/anhydrides and azo derivatives. The L-Threoninederivatives are capable of forming a wide variety of pharmaceuticallyacceptable salts with various inorganic and organic acids. These acidsthat may be used to prepare pharmaceutically acceptable acid additionsalts of such basic compounds are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitride,sulfate, bisulfate, phosphate, formate, acetate, citrate, tartate,lactate, and the like.

As indicated herein, in one embodiment, the present invention isdirected to a derivative wherein the derivative comprises a drug, e.g.,Ibuprofen and the amino acid L-Threonine esterified to the POOH group ofIbuprofen. This results in a derivative where L-Threonine is attached tothe Ibuprofen, for example, by a covalent bond.

The compounds of the present invention are prepared by art recognizedtechniques. For examples, if the drug contains an OH group, said ashydroxychloroquine, then the carboxyl group of L-Threonine is reacted toform a covalent bond, resulting in an ester or the hydroxy group may beoxidized to an acid and the covalent bond may be formed between thecarboxy group thus formed and the OH group of the hydroxychloroquine.The former, however, is preferred. Alternatively, as describedhereinabove, if the drug has an amino group thereon, then theL-Threonine may be reacted through the carboxy group of the Threoninewith the drug under amide forming conditions to form an amide as thecovalent bond, or the hydroxy group of the side chain may be oxidized toa carboxy group and the amide bond is through the carboxy group of theoxidized hydroxy group of the side chain, although, the former ispreferred. Alternatively, if the drug has a carboxy group or acylatingderivative thereon, it may be reacted with the amino group of theL-Threonine to form an amide under amide forming conditions or the OHgroup of the side chain of Threonine may be converted to the NH₂ groupand this amino group may form an amide bond with the carboxy group oracylating derivative of the drug, but again the former is preferred.Additionally if the drug has a carboxy group thereon, the hydroxy groupof the side chain of the L-Threonine may be reacted with the carboxygroup or acylating derivative, thereof under esterification conditionsto form the ester linkage between the L-Threonine and the drug, asdescribed hereinabove.

If any portion of the L-Threonine group or the drug is reactive underthe reaction conditions, it is protected by a protecting group known inthe art. After the completion of the reaction, the protecting group isremoved. Examples of protecting groups that could be used are describedin the book entitled, “Protective Group in Organic Synthesis” byTheodora W. Greene, John Wiley & Sons, 1981, the contents of which areincorporated by reference.

For example, if L-Threonine is to be reacted with a drug containing COOHgroup to form the ester, then the COOH group of L-Threonine requiresprotection using protecting groups known in the art. Examples ofsuitable protecting groups can be esters, such as cyclohexyl esters,t-butyl esters, benzyl esters, allyl esters, 9-fluorophenyl-methylgroups or adamantyl groups, such as 1- or 2-adamantyl which can beremoved after the esterfication reaction is completed using techniquesknown to one of ordinary skill in the art.

If the L-Threonines hydroxyl group is to be protected in a reactionbetween L-Threonine and a drug containing OH group, then the OH group ofL-Threonine is protected with protecting groups known in the art, e.g.,ethers, such as benzyl ether or t-butyl ether. Removal of the benzylether can be effected by liquid hydrogen fluoride, while the t-butylether can be removed by treatment with trifluoroacetic acid. Suitableprotecting groups for phenolic side chain groups can be ethers, asabove, including benzyl or t-butyl ether or 2,6-dichlorobenzyl,2-bromobenzyloxycarbonyl, 2,4-dintrophenyl and the like.

Moreover, the products prepared by the present invention can be purifiedto be made substantially pure by techniques known to one of ordinaryskill in the art, such as by chromatography, e.g., HPLC, crystallizationand the like. By substantially “pure” it is meant that the productcontains no more than about 10% impurity therein (w/w). Further, theproducts can be prepared to be made substantially sterochemically pureusing techniques known to one of ordinary skill in the art. By“stereochemically pure” it is meant that the product contains no morethan 10% by weight of the enantiomer, disasteromer or otherstereoisomer.

The drugs to which the L-Threonine moiety is covalently bonded(hereinafter “derivatives of the present invention”), or pharmaceuticalacceptable salts, pharmaceutical acceptable solvates, esters,enantiomers, diastereomers, N-Oxides, polymorphs, and the like, asdescribed herein, can be made into pharmaceutical compositions alongwith a pharmaceutical acceptable carrier, and optionally but desirablypharmaceutically acceptable excipients using techniques known to one ofordinary skill in the art.

The pharmaceutical compositions of the present invention are used intherapeutically effective amounts.

The physician will determine the dosage of the derivatives of thepresent invention which will be most suitable and it will vary with theform of administration and the particular compound chosen, andfurthermore, it will vary depending upon various factors, including butnot limited to, the patient under treatment and the age of the patient,the severity of the condition being treated and the like and theidentity of the derivative of the present invention administered. Hewill generally wish to initiate treatment with small dosagessubstantially less than the optimum dose of the derivative of thepresent invention and increase the dosage by small increments until theoptimum effect under the circumstances is reached. It will generally befound that when the composition is administered orally, largerquantities of the active agent will be required to produce the sameeffect as a smaller quantity given parenterally. The compounds of thepresent invention are useful in the same manner as the correspondingdrug from which they are prepared, and the dosage level is preferably ofthe same order of magnitude as is generally employed with these othertherapeutic agents. When given parenterally, the compounds areadministered generally in dosages of, for example, about 0.00001 toabout 10,000 mg/kg/day, also depending upon the host and the severity ofthe condition being treated and the compound of the present inventionutilized.

In a preferred embodiment, the compounds of the present inventionutilized are orally administered in amounts ranging from about 0.0001 mgto about 1000 mg per kilogram of body weight per day, depending upon theparticular mammalian host or the disease to be treated, more preferablyfrom about 0.01 to about 500 mg/kg body weight per day. This dosageregimen may be adjusted by the physician to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The derivative of the present invention may be administered in anyconvenient manner, such as by oral, intravenous, intramuscular orsubcutaneous, transdermal, rectal, vaginal, buccal, nasal routes and thelike.

The derivative of the present invention may be orally administered, forexample, with an inert diluent or with an assimilable edible carrier, orit may be enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly into thefood of the diet. For oral therapeutic administration, the derivative ofthe present invention may be incorporated with excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. Such compositionsand preparations should contain at least 0.0001% of the derivative ofthe present invention. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 0.001 to about 99.9999% of the weight of the unit. The amount ofthe derivative of the present invention used in such therapeuticcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention containbetween about 0.0001 mg and about 4000 gm of derivative. The tablets,troches, pills, capsules and the like may also contain the following: Abinder such as gum tragacanth, acacia, corn starch or gelatin;excipients such as dicalcium phosphate; a disintegrating agent such ascorn starch, potato starch, alginic acid and the like; a lubricant suchas magnesium stearate; and a sweetening agent such as sucrose, lactoseor saccharin may be added or a flavoring agent such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier.

Various other materials may be present as coatings or otherwise modifythe physical form of the dosage unit. For instance, tablets, pills, orcapsules may be coated with shellac, sugar or both. A syrup or elixirmay contain the compound of the present invention, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, thecompound of the present invention may be incorporated intosustained-release preparations and formulations. For example, sustainedrelease dosage forms are contemplated wherein the compound of thepresent invention is bound to an ion exchange resin which, optionally,can be coated with a diffusion barrier coating to modify the releaseproperties of the resin or wherein the compound of the present inventionis associated with a sustained release polymer known in the art, such ashydroxypropylmethylcellulose and the like.

The compound of the present invention may also be administeredparenterally or intraperitoneally. It is especially advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, e.g., PEG 100, PEG200, PEG 300, PEG 400, and the like, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the form is usually sterile andmust be fluid to the extent that syringability exists. It must be stableunder the conditions of manufacture and storage and usually must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and one or more liquid polyethylene glycol, e.g. asdisclosed herein and the like), suitable mixtures thereof, and vegetableoils. The proper fluidity can be maintained, for example, by the use ofa coating such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the compoundof the present invention in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the sterilized compound of the presentinvention into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders, the above solutions are vacuum dried orfreeze-dried, as necessary.

The compound of the present invention can also be applied topically, ase.g., through a patch using techniques known to one of ordinary skill inthe art. It can be administered buccally by preparing a suitableformulation thereof and utilizing procedures well known to those skilledin the art. These formulations are prepared with suitable non-toxicpharmaceutically acceptable ingredients. These ingredients are known tothose skilled in the preparation of buccal dosage forms. Some of theseingredients can be found in Remington's Pharmaceutical Sciences, 17^(th)edition, 1985, a standard reference in the field. The choice of suitablecarriers is highly dependent upon the exact nature of the buccal dosageform desired, e.g., tablets, lozenges, gels, patches and the like. Allof these buccal dosage forms are contemplated to be within the scope ofthe present invention and they are formulated in a conventional manner.

The formulation of the pharmaceutical compositions of the presentinvention may be prepared using conventional methods using one or morephysiologically and/or pharmaceutically acceptable carriers orexcipients. Thus, the compounds of the present invention and theirpharmaceutically acceptable salts and solvates may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, buccal, parenteral, or rectal administration. Fororal administration, the pharmaceutical compositions may take the formof, for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (forexample, pregelatinized maize starch, polyvinylpyrrolidone, orhydroxypropylmethyl cellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc, or silica); disintegrants (forexample, potato starch, or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulfate). The tablets may be coated bymethods well known in the art.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups, or suspensions, or they may be presented asa dry product for constitution with water or other suitable vehiclesbefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives, such as suspendingagents (for example, sorbitol syrup, corn syrup, cellulose derivativesor hydrogenated edible oils and fats); emulsifying agents (for example,lecithin or acacia); non-aqueous vehicles (for example, almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(for example, methyl or propyl p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate. Preparations for oral administrationmay be suitably formulated to give controlled release of the activederivative.

The derivative of the present invention may be formulated for parenteraladministration by injection, for example, by bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form, for example, in ampoules, or in multi-dose containers, withan added preservative. The compositions of the present invention maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the derivative ofthe present invention may be in the powder form for constitution with asuitable vehicle, for example, sterile pyrogen-free water, before use.

The derivatives of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, forexample, containing conventional suppository bases such as cocoa butteror other glycerides.

In addition to the formulations described previously, the derivative ofthe present invention may also be formulated as a depot preparation.Such long acting formulations may be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the derivatives of the present inventionmay be formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions containing the derivatives of thepresent invention may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredients. The pack may for example comprise metal or plasticfoil, such as blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

In tablet form, it is desirable to include a lubricant which facilitatesthe process of manufacturing the dosage units; lubricants may alsooptimize erosion rate and drug flux. If a lubricant is present, it willbe present on the order of 0.01 wt. % to about 2 wt. %, preferably about0.01 wt. % to 0.5 wt, %, of the dosage unit. Suitable lubricantsinclude, but are not limited to, magnesium stearate, calcium stearate,stearic acid, sodium stearylfumarate, talc, hydrogenated vegetable oilsand polyethylene glycol. As will be appreciated by those skilled in theart, however, modulating the particle size of the components in thedosage unit and/or the density of the unit can provide a similareffect—i.e., improved manufacturability and optimization of erosion rateand drug flux—without addition of a lubricant.

Other components may also optionally be incorporated into the dosageunit. Such additional optional components include, for example, one ormore disintegrants, diluents, binders, enhancers, or the like. Examplesof disintegrants that may be used include, but are not limited to,crosslinked polyvinylpyrrolidones, such as crospovidone (e.g.,Polyplasdone® XL, which may be obtained from GAF), cross-linkedcarboxylic methylcelluloses, such as croscanmelose (e.g., Ac-di-sol®,which may be obtained from FMC), alginic acid, and sodium carboxymethylstarches (e.g., Explotab®, which may be obtained from Edward Medell Co.,Inc.), agar bentonite and alginic acid. Suitable diluents are thosewhich are generally useful in pharmaceutical formulations prepared usingcompression techniques, e.g., dicalcium phosphate dihydrate (e.g.,Di-Tab®, which may be obtained from Stauffer), sugars that have beenprocessed by crystallization with dextrin (e.g., co-crystallized sucroseand dextrin such as Di-Pak®, which may be obtained from Amstar), calciumphosphate, cellulose, kaolin, mannitol, sodium chloride, dry starch,powdered sugar and the like. Binders, if used, are those that enhanceadhesion. Examples of such binders include, but are not limited to,starch, gelatin and sugars such as sucrose, dextrose, molasses, andlactose. Permeation enhancers may also be present in the novel dosageunits in order to increase the rate at which the active agents passthrough the buccal mucosa. Examples of permeation enhancers include, butare not limited to, dimethylsulfoxide (“DMSO”), dimethyl formamide(“DMF”), N,N-dimethylacetamide (“DMA”), decylmethylsulfoxide (“C₁₀MSO”),polyethylene glycol monolaurate (“PEGML”), glycerol monolaurate,lecithin, the 1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (available under the trademarkAzone™ from Nelson Research & Development Co., Irvine, Calif.), loweralkanols (e.g., ethanol), SEPA® (available from Macrochem Co.,Lexington, Mass.), cholic acid, taurocholic acid, bile salt typeenhancers, and surfactants such as Tergitol®, Nonoxynol-9® andTWEEN-80®.

Flavorings may be optionally included in the various pharmaceuticalformulations. Any suitable flavoring may be used, e.g., mannitol,lactose or artificial sweeteners such as aspartame. Coloring agents maybe added, although again, such agents are not required. Examples ofcoloring agents include any of the water soluble FD&C dyes, mixtures ofthe same, or their corresponding lakes.

In addition, if desired, the present dosage units may be formulated withone or more preservatives or bacteriostatic agents, e.g., methylhydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkoniumchloride, or the like.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents for pharmaceuticalactive substances well known in the art. Except insofar as anyconventional media or agent is incompatible with the derivative, theiruse in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of derivative calculated to producethe desired therapeutic effect in association with the requiredpharmaceutical carrier.

The derivative of the present invention is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedescribed. A unit dosage, for example, contains the compound of thepresent invention in effective amounts. For example, in a preferredembodiment, the amounts range from about 10 mg, e.g. in humans, or aslow as 1 mg (for small animals) to about 2000 mg. If placed in solution,the concentration of the derivative of the present invention preferablyranges from about 10 mg/mL to about 250 mg/mL. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients. In the case of buccaladministration, the derivatives of the present invention are preferablyin the buccal unit dosage form present in an amount ranging from about10 to about 50 mg.

The derivatives of the present invention are effective in treatingdisease or conditions in which the corresponding drug (without theL-Threonine derivative of the present invention) normally are used.

As used herein the term “treating” refers to reversing, alleviating orinhibiting the progress of a disease, disorder or condition, or one ormore symptoms of such disease, disorder or condition, to which such termapplies. As used herein, “treating” may also refer to decreasing theprobability or incidence of the occurrence of a disease, disorder orcondition in a mammal as compared to an untreated control population, oras compared to the same mammal prior to treatment. For example, as usedherein, “treating” may refer to preventing a disease, disorder orcondition, and may include delaying or preventing the onset of adisease, disorder or condition, or delaying or preventing the symptomsassociated with a disease, disorder or condition. As used herein,“treating” may also refer to reducing the severity of a disease,disorder or condition or symptoms associated with such disease, disorderor condition prior to a mammal's affliction with the disease, disorderor condition. Such prevention or reduction of the severity of a disease,disorder or condition prior to affliction relates to the administrationof the composition of the present invention, as described herein, to asubject that is not at the time of administration afflicted with thedisease, disorder or condition. As used herein “treating” may also referto preventing the recurrence of a disease, disorder or condition or ofone or more symptoms associated with such disease, disorder orcondition. The terms “treatment” and “therapeutically,” as used herein,refer to the act of treating, as “treating” is defined above.

Prophylaxis or preventing or any like term, refers to decreasing theprobability or incidence of the occurrence of a disease, disorder orcondition in a mammal. It also includes delaying or preventing the onsetof a disease, disorder or condition or delaying the symptoms associatedwith a disease, disorder or condition. In addition, it also refers toretarding the occurrence of a disease, disorder or condition in amammal.

As used herein the term “patient” or “subject” refers to a warm bloodedanimal, and preferably mammals, such as, for example, cats, dogs,horses, cows, pigs, mice, rats and primates, including humans. Thepreferred patient is humans.

The derivatives of the present invention exhibit the same utility as thecorresponding drug without the L-Threonine linkage. The derivativeexhibits an enhanced therapeutic quality. That is, they exhibit at leastone and more preferably at least two enhanced therapeutic qualitiesrelative to the drug which has not been transformed to the derivative ofthe present invention prior to administration. These include, but arenot limited to

-   -   a. Improved taste, smell    -   b. Desired octanol/water partition coefficient (i.e., solubility        in water/fat) The L-Threonine when covalently bound to a drug        may enhance solubility in water or it may enhance absorption by        the mammal to which it is administered.    -   c. Improved stability in-vitro and in-vivo    -   d. Enhanced penetration of blood-brain barrier    -   e. Elimination of first-pass effect in liver, i.e., the drug is        not metabolized in the liver and therefore more drug in system        circulation    -   f. Reduction of entero-hepatic recirculation (this improves        bio-availability)    -   g. Painless injections with parenteral formulations    -   h. Improved bio-availability    -   i. Improved changes in the rate of absorption (increase vs lack        thereof)    -   j. Reduced side effects    -   k. Dose proportionality    -   l. Selective hydrolysis of the derivative at site of action    -   m. Controlled release properties    -   n. Targeted drug delivery    -   o. Reduction in toxicity, hence, improved therapeutic ratio    -   p. Reduced dose    -   q. Alteration of metabolic pathway to deliver more drug at the        site of action    -   r. Increased solubility in aqueous solution    -   s. Enhanced efficacy    -   t. Separation of enantiomers and diasteroisomers

A dose proportionality claim requires that when the drug is administeredin escalating doses, proportionally escalating amounts of active drug isdelivered into the blood stream. This is measured by determining thearea under the plasma concentration vs. time curve obtained afteradministering a drug via any route other than IV route and measuring thesame in plasma/blood. A simple mathematical procedure is as follows: Forexample, a drug is administered at e.g., 3 different doses, 10, 100 and1000 mg, orally to a patient, the area under the plasma concentrationtime curve (AUC) is measured. Then each total AUC is divided by thedose, and the result should be the same for all three doses. If it isthe case, then there is dose proportionality. Lack of doseproportionality indicates any one or more of thepharmacokinetic/pharmacological mechanisms are saturable, includingabsorption, metabolism or the number of receptor sites available forpharmacological response.

For example in the above study, assume the AUC values of 100, 1000 and10,000 are obtained, in this case the dose proportionality isinappropriate. When there is lack of dose proportionality, there iseither more or less amount of drug in the plasma, depending upon whichmechanism is saturable. The following are the possibilities: SaturableAbsorption. If this is the case, as the dose is increased,proportionally less and less of the drug is absorbed, hence overall AUCwill decrease as the dose is increased.

Saturable metabolism of elimination. If this is the case, then more andmore of the drug will be circulating in the blood, and the AUC willincrease with increasing dose. Saturable pharmacological receptor sites:In this case, since all the receptor sites will eventually be occupiedby the drug, any additional drug will not increase the response, butmost likely increase the toxicity of the drug/derivative. Thus,increasing dose will not result in increasing response and in fact mayreduce the therapeutic index.

Dose proportionality is an excellent response profile, since one canpredict accurately the pharmacological response and curative power atall doses. Thus dose proportionality is a desirable quality for anydrug. Furthermore, achievement of dose proportionality is also dependentupon the formulation, and fed/fasted differences.

Thus, various dosage forms are available of drugs for which theL-Threonine is bound and they are prepared by conventional methods.These various dosage forms include:

-   -   i. Oral liquid dosage (Controlled release and immediate release        liquids containing sugar and sugar free, dye and dye free,        alcohol and alcohol free formulations, including chewable        tablets)    -   ii. Oral solid dosage (Controlled release and immediate release        tablets, capsules and caplets    -   iii. Intravenous (Injections, both ready to use and lyophilized        powders)    -   iv. Intramuscular (Injections, both ready to use and lyophilized        powders)    -   v. Subcutaneous (Injections, both ready to use and lyophilized        powders)    -   vi. Transdermal (Mainly patches)    -   vii. Nasal (Sprays, formulations for nebulizer treatments)    -   viii. Topical (Creams, ointments)    -   ix. Rectal (Creams, ointments and suppositories)    -   x. Vaginal (Creams, ointments and pessaries)    -   xi. Ocular (Drops and ointments)    -   xii. Buccal (Chewable and now chewable tables)

Many drugs discussed herein, especially in the table hereinbelow arecharacteristically highly hydrophobic and readily precipitate in thepresence of even very minor amounts of water, e.g., on contact with thebody (e.g., stomach fluids). It is accordingly extremely difficult toprovide e.g., oral formulations which are acceptable to the patient interms of form and taste, which are stable on storage and which can beadministered on a regular basis to provide suitable and controllingpatient dosing.

Proposed liquid formulations, e.g., for oral administration of a numberof drugs shown herein in the table have heretofore been based primarilyon the use of ethanol and oils or similar excipient as carrier media.Thus, the commercially available drink-solutions of a number of drugsemploy ethanol and olive oil or corn oil as carrier medium inconjunction with solvent systems comprising e.g., ethanol and LABRIFILand equivalent excipient as carrier media. Use of the drink solution andsimilar composition as proposed in the art is, however, accompanied by avariety of difficulties.

Further, the palatability of the known oil based system has provedproblematic. The taste of the known drink-solution of several drugs is,in particular, unpleasant. Admixture with an appropriate flavored drink,for example, chocolate drink preparation, at high dilution immediatelyprior to ingestion has generally been practiced in order to make regulartherapy at all acceptable. Adoption of oil-based systems has alsorequired the use of high ethanol concentrations which is itselfinherently undesirable, in particular where administration to childrenis foreseen. In addition, evaporation of the ethanol, e.g., fromcapsules (adopted in large part, to meet problems of palatability, asdiscussed or other forms (e.g., when opened)) results in the developmentof drug precipitates. Where such compositions are presented in, forexample, soft gelatin encapsulated form, the encapsulated product ispackaged in an air-tight component, for example, an air-tight blister oraluminum-foil blister package. This in turn renders the product bothbulky and more expensive to produce. The storage characteristics of theaforesaid formulations are, in addition, far from ideal.

Bioavailability levels achieved using existing oral dosage system for anumber of drugs are also low and exhibit wide variation betweenindividuals, individual patient types and even for single individuals atdifferent times during the course of therapy. Reports in the literatureindicate that currently available therapy employing the commerciallyavailable drug drink solution provides an average absolutebioavailability of approximately 10-30% only, with the marked variationbetween individual groups, e.g., between liver (relatively lowbioavailability) and bone-marrow (relatively high bioavailability)transplant recipients. Reported variation in bioavailability betweensubjects has varied from one or a few percent for some patients, to asmuch as 90% or more for others. And as already noted, marked change inbioavailability for individuals with time is frequently observed. Thus,there is a need for a more uniform and high bioavailability of a numberdrugs shown herein in patients.

Use of such dosage forms is also characterized by extreme variation inrequired patient dosing. To achieve effective therapy, drug blood orblood serum levels have to be maintained within a specified range. Thisrequired range can, in turn, vary, depending on the particular conditionbeing treated, e.g., whether therapy is to prevent one or morepharmacological actions of a specific drug and when alternative therapyis employed concomitantly with principal therapy. Because of the widevariations in bioavailability levels achieved with conventional dosageforms, daily dosages needed to achieve required blood serum levels willalso vary considerably from individual to individual and even for asingle individual. For this reason it may be necessary to monitorblood/blood-serum levels of patients receiving drug therapy at regularand frequent intervals. This is inevitably time consuming andinconvenient and adds substantially to the overall cost of therapy.

It is also the case that blood/blood serum levels of a number of drugsusing available dosage systems exhibit extreme variation between peakand trough levels. That is, for each patient, effective drug levels inthe blood vary widely between administrations of individual dosages.

Beyond all these very evident practical difficulties lies the occurrenceof undesirable side reactions already alluded to, observed employingavailable oral dosage forms.

Several proposals to meet these various problems have been suggested inthe art, including both solid and liquid oral dosage forms. Anoverriding difficulty which has however remained is the inherentinsolubility of the several of the drugs without the Threonine attachedthereto in aqueous media, hence preventing the use of a dosage formwhich can contain the drugs in sufficiently high concentration to permitconvenient use and yet meet the required criteria in terms ofbioavailability, e.g. enabling effective absorption from the stomach orgut lumen and achievement of consistent and appropriately highblood/blood-serum levels.

However, the compounds of the present inventions overcome the problemsenumerated hereinabove.

For example, the derivative of the present invention significantlyenhances the solubility of the drug from which it is synthesized inaqueous solutions relative to the non-derivative form of thepharmaceutical, thereby avoiding the need to utilize a carrier, such asethanol or castor oil when administered as a solution. Moreover, thederivatives of the present invention do not exhibit the side effects ofthe prior art formulations. Further, it has been found that when many ofthe drugs are administered covalently bound to threonine, in accordancewith the present invention, there is enhanced oral absorption, therebyenhancing significantly its bioavailability and its efficacy.

The utility of the derivative is the same as the corresponding drug(without the L-Threonine moiety attached). The utiluity is described inthe literature such as the Physicians Desk Reference, 2004 edition, thecontents of which are incorporated by reference.

Enantiomeric or Stereoisomeric Separation Using L-Threonine

In a separate emobodiment, it has been surprisingly found thatL-Threonine can separate racemic mixtures of drug molecules into theirpure enantiomers. Normally such separations are effected with heroicmethods of synthesis, chromatography, fermentation with microbes, orusing special bioenzymes. However, the present inventor has found thatwhen the racemate drug was reacted with L-Threonine to form an ester,the two enantiomeric esters had different physicochemicalcharacteristics. For example, one enantiomeric ester was more soluble inwater and vice versa, hence the inventor could readily separate the twoenantiomeric L-threonine drug esters.

Even more surprisingly, the inventor noted that only L-Threonine canseparate the racemic mixtures of the drugs, and similar OH containingother amino acids such as L-Serine, L-hydroxyproline or otherhydroxylamino acids did not have the same effectiveness as L-Threonine.Other than L-Threonine, none of the other hydroxylamino acids werecapable of readily separating the drug enantiomers to the same degreewith minimal effort.

It was even more surprising, that when an ester of L-Threonine was madewith a drug containing either OH or COOH group, the resulting ester notonly resolved the chiral centers, but also the resulting drug ester wasmore effective in almost all cases than the intact drug. This wasevident with racemic drugs such as ibupronfen and Ketorolac, andnon-racemic drugs such as Aspirin, enalapril and Fenofibric acid.

Furthermore, it was highly surprising, that the resulting L-Threonineester of the drugs did not act as a prodrug. One who is reasonablytrained in the art for forming prodrugs and prodrug esters would havethought that an ester would be a prodrug and will release the activedrug during its transport through the body, either at the site of oralabsorption, GI wall, blood, liver or other organs of the body. But tothe inventor's surprise, in many cases, there was no active drug in theblood stream after oral administration of the L-Threonine ester of thedrug. In other words, the derivative of the present invention in whichthe L-Threonine is covalently bound to the drug, in many cases, are notprodrugs.

For example, the inventor noted that in case of Ibuprofen and AspirinThreonine Esters, there was practially no Ibuprofen or Aspirin found inthe blood stream of human subjects after oral administration of therespective ester. Surprisingly, the inventor noted that, not only wasthe parent drug not being released in the blood stream, but also theThreonine ester of the active drug exhibited better efficacy than theparent drug in all animal models and human trials.

It was also quite surprising that improved efficacy and enantiomericseparation with Threonine was non-specific to the drugs to whichL-Threonine was attached. For example, drugs with quite widely varyingchemical structures with differing polarities, molecular weights,therapeutic categories, molecular formula, active groups, active siteand active moiety, and drugs with highly dissimilar drug-receptortopography were able to react with L-Threonine and resulted in improvedtherapeutic index.

Moreover, the inventor has also found that the drug to which theThreonine was bound is less toxic. For example, Ibuprofen racemicmixture was more toxic to rats under chronic administration. 40% of therats receiving Ibuprofen racemic mixture died during the 28-day chronictoxicity study. However, all the animals receiving L-Threonine ester ofIbuprofen survived and appeared healthy in the same parallel trial, buthad somewhat reduced body weight.

Similar results in toxicity were seen with Aspirin. When the L-Threonineester of Aspirin was administered orally to rats, no GI side effectswere seen, however, with intact aspirin, severe GI anatomical changes,bleeding, ulcers and other side effects were noted in the same group ofrats.

While the L-Threonine ester had better pharmacological profile than theparent drug, and it was able to separate the racemic mixtures into theirpure enatitomers, the inventor surprisingly found that such effects wererepeated again and again with various other drugs.

For example, the inventor found that L-Threonine also separatedKetoprofen, while none of the other OH containing amino acids such asHydroxyproline or Serine were unable to separate the enantiomers asreadily as Threonine did. Without employing any heroic methods, theseparation of the enantiomers of the L-Threonine ester of Ketoprofen waseasily achieved. Furthermore, the inventor also noted that L-Threonineseparated the racemic mixture of Ketorolac into its enantiomerically andstereochemically pure isomers as well.

Thus, L-threonine is an important agent in the separation of enantiomersand diaseteroisomers. It was possible to simply hydrolyze theL-threonine ester of Ibuprofen into either S(+) or R(−) ibuprofen,rather readily with usual organic reaction methods. Similarly theinventor was able to hydrolyze the L-Threonine esters of Ketorprofen andKetorolac to release the pure enantiomers.

Moreover, the inventor has found that the resulting L-Threonine estersof the individual enantiomers are more active, and less toxic, resultingin improved therapeutic efficacy.

Such improved therapeutic efficacy is not only related to isolating theright isomer, but the inventor has also found that this is true in caseof other drugs that are not racemates or stereoisomers. For example, theinventor noted that non-racemates such as Aspirin, Fenofibric Acid,enalaprilat and other drugs were also of high therapeutic index orgreater effiacy, when L-Threonine esters were formed with these drugs.The use of L-threonine was able to separate molecules of widely varyingand dissimilar organic chemical structures. In those cases where issueof separation of enantiomers does not arise, derivatives of the presentinvention e.g., L-Threonine esters of achiral drugs showed bettertherapeutic qualities compared to their parent drugs.

Therefore the derivatives of the present invention for a number of drugsare not prodrugs, have intact activity, better efficacy and lesstoxicity, resulting in improved therapeutic index.

With respect to hydroxychloroquine, one enantiomer is active againstplasmodium vivax parasite and the other isomer is active for its diseasemodifying effect for rheumatoid arthritis. However, it is postulatedthat only one of these isomer is responsible for optical retinopathy.Hence by administereing the less toxic variety, by covalently bondingthe L-Threonine moiety thereto by an ester linkage between the OH groupof the hydroxychloroquine and the carboxy gropu of the Threonine theoptical toxicity of hydroxychlorqouine is significantly reduced.

As an additional specific example of usefulness of L-threonine basedseparation of racemic mixture can be applied to carvedilol, with thefollowing structure:

which is(±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol.Carvedilol is a racemic mixture in which nonselective β-adrenoreceptorblocking activity is present in the S(−) enantiomer and α-adrenergicblocking activity is present in both R(+) and S(−) enantiomers at equalpotency. Carvedilol has no intrinsic sympathomimetic activity. However,this drug can be readily separated into its pure isomers, usingL-threonine, by reacting the OH group with COOH of L-Threonine. Thisachieves the following results:

-   -   a) When specific beta-adrenergic blocking action is required to        reduce the blood pressure, one could administer        S(−)Carvedilol-L-threonine ester.    -   b) When specific alpha-1 adrenergic blocking activity is needed,        it is preferable to administer R(+)Carvedilol-L-threonine ester.        This is due to the fact that 3 times higher levels of R(+) is        available in human plasma after oral administration compared to        the S(−) variety due to first pass metabolism.

The following non-limiting examples further illustrate the invention:

Pure Enantiomeric L-Threonine Esters of Non-Steroidal Anti-InflammatoryDrugs (NSAIDs)

The NSAIDs comprise a class of structurally distinctive, carboxylic acidmoiety attached to a planar aromatic functionality, Examples include:acetyl salicyclic acid, salicyclic acid, diflunisal, ibuprofen,fenoprofen, carprofen, flurbiprofen, ketoprofen, naproxen, sulindac,indomethacin, etodolac, tolmetin, ketorolac, diclofenac, andmeclofenamate, and the like. The NSADIs posess anti-inflammatory,analgesic, antipyretic and anti-clotting activity.

Examples of the chemical structures of this unique class of compoundsshowing wide variety of pharmacological activities are shown below.

NSAIDs are widely used for the treatment of acute and chronic pain,management of edema, tissue damage resulting from inflammatory jointdiseases and also, effective anti-clotting agents in the treatment ofmyocardial infraction. A number of the agents also possess antipyreticactivity in addition to analgesic and anti-inflammatory action, thususeful in reducing fever.

Some drugs in the above group have also been prescribed for RheumatoidArthritis, Osteoarthritis, acute gout, ankolysing spondylitis, anddysmenorrhea.

Mechanism of Action:

The major mechanism by which the NSAIDs produce their therapeutic effectis via inhibition of prostaglandin synthesis. Specifically NSAIDsinhibit cyclooxygenases, such as COX-1 and COX-2 enzymes, where thesetwo enzymes are responsible for synthesis of prostaglandins. While COX-1enzyme is important for the regulation of platelet aggregation,regulation of blood flow in kidney and stomach, and regulation ofgastric acid secretion, COX-2 enzyme plays an important role in the painand inflammatory processes. NSAIDs significantly increase clotting timeand can be used for the prophylaxis of thromboembolism and myocardialinfarction.

All NSAIDs are relatively medium to strong organic acids with pKa's inthe 3-6 range. Most of them are carboxylic acid derivatives. An acidicgroup is essential for COX inhibitory activity and in physiological pH,all the NSAIDs are ionized. All of them have quite varying hydrophiliclipophilic balance, and these are functions of their aryl, aromatic andaliphatic side chains and other heterocyclic variations in theirstructures. Most of the NSAIDs are highly bound to plasma proteins andoften competitively replace other drugs which have similar affinity forplasma proteins. Hence concomitant administration of NSAIDs with anotherdrug, e.g. another therapeutic class, must be carefully evaluated toprevent drug interactions. Most of the drugs, due to acidic carboxylgroup are metabolized by the mammals via conjugation. The major pathwayof metabolic clearance of a number of NSAIDs is glucuronidation followedby renal elimination.

Use of acetylsalicylic acid (aspirin) in the prophylaxis of coronaryheart diseases is now well known, and this drug has proved to be alifesaver for a number of patients with myocardial infarction. Severaladditional uses have already been documented for aspirin, for example,it was recently reported in the medical journal Lancet (Vol 349, p 1641)that aspirin reduces the risk of stroke in patients with early warningsigns of transient ischemic heart attacks. Pre-eclampsia and fetalgrowth retardation, both caused by blockages of the blood vessels of theplacenta, are two of the commonest complications of pregnancy—there aremillions of cases of pre-eclampsia in the world each year. In a trialinvolving more than 9000 women in 16 countries, a daily dose of 60 mgaspirin reduced the risk of pre-eclampsia by 13 per cent. (AspirinFoundation website). Aspirin has also been shown to be effective in somestudies to prevent colon cancer, lung cancer and pancreatic cancer inpost-menopausal women. Since aspirin can improve blood flow, itsusefulness in the treatment of diabetes and certain forms of dementiasuch as Alzheimer's disease are becoming increasingly clear.

Because of their unique pharmaceutical potential, the NSAIDs haveattracted considerable attention in the press. The primary area ofclinical investigation for these drugs has been as non-steroidalanti-inflammatory agents, in particular in relation to their applicationto patients suffering from pain, arthritis (Rheumatoid and Osteo), otherinflammatory reactions, and fever and for the prophylaxis of coronaryheart diseases. These drugs are also used in the treatment of migraineheadache, menstrual syndromes, back pain and gout.

Even more troubling health care issues recently become known for theCOX-2 inhibitors such as celecoxib, rofecoxib and valdecoxib which wereremoved from the world market for causing poor cardiovascular health,leading to heart attack and other problems. In light of these newdevelopments, the reversible inhibition of platelet aggregation by theL-threonine ester of NSAIDs of the present invention offers bettertherapy for patients who suffer from the aforementioned maladres, e.g.,rheumatoid and osetoarthritis.

Despite the very major contribution which NSAIDs have made, difficultieshave been encountered in providing more effective and convenient meansof administration (e.g., galenic formulations, for example, oral dosageform, which are both convenient and for the patient as well as providingappropriate bioavailability and allowing dosaging at an appropriate andcontrolled dosage rate) as well as the reported occurrence ofundesirable side reactions; in particular severe gastric and duodenalulcers, mucosal erythema, and edema, erosions, perforations, blood instool, and ulcerative colitis have are obvious serious impediments totheir wider use or application. The dual injury theory involvesNSAID-mediated direct damage, followed by a systemic effect in whichprostaglandin synthesis is inhibited. Topical injury may also occur as aresult of the biliary excretion of active hepatic metabolites andsubsequent duodenogastric reflux. (Arthritis and Rheumatism 1995;38(1):5-18) The effects are additive; either topical or systemicmechanisms alone are sufficient to produce gastro duodenal mucosaldamage.

Moreover, the above mentioned NSAIDs, (without being bond toL-Threonine) are characteristically highly hydrophobic and readilyprecipitate in the presence of even very minor amounts of water, e.g.,on contact with the body (e.g., stomach fluids). It is accordinglyextremely difficult to provide e.g., oral formulations which areacceptable to the patient in terms of form and taste, which are stableon storage and which can be administered on a regular basis to providesuitable and controlling patient dosaging.

Proposed liquid formulations, e.g., for oral administration of NSAIDs,have heretofore been based primarily on the use of natural gums, likeXanthan, cellulose, citric acid, and lime flavor etc. See e.g., U.S.Pat. No. 5,780,046. Commercially available NSAIDs drink-solution employsincompatible orange color and berry flavor, citric acid, Xanthan Gum,polysorbate 80, pregelatinized starch, glycerin, sodium benzoate, andadditional artificial colors and flavors. Use of the drink solution andsimilar composition as proposed in the art is however accompanied by avariety of difficulties.

Further, the palatability of the known oil based system has provedproblematic. The taste of the known drink-solution is, in particular,unpleasant. Admixture with an appropriate flavored drink, for example,chocolate drink preparation, at high dilution immediately prior toingestion has generally been practiced in order to make regular therapyat all acceptable. Adoption of oil based systems has also required theuse of high ethanol concentrations which, in and of itself is inherentlyundesirable, in particular where administration to children is forseen.In addition, evaporation of the ethanol, e.g., from capsules (adopted inlarge part, to meet problems of palatability, as discussed or otherforms (e.g., when opened)) results in the development of a NSAIDprecipitate. Where such compositions are presented in, for example, softgelatin encapsulated form, this necessitates packaging of theencapsulated product in an air-tight component, for example, anair-tight blister or aluminum-foil blister package. This in turn rendersthe product both bulky and more expensive to produce. The storagecharacteristics of the aforesaid formulations are, in addition, far fromideal.

Gastric irritability of the NSAIDs has been a topic of great concern tothe practicing physicians and as well as patients. Acute uses ofaspirin, fenoprofen, flurbiprofen, indomethacin, ketorolac,meclofenamate, mefanamic acid, and piroxicam produce serious GI sideeffects. Even Ibuprofen is shown to cause severe gastric lesions uponlong term use. Gastrointestinal toxicity is the most frequentlyencountered side effect associated with NSAIDs and presents considerableconcern. Approximately one half of all hospital admissions for ableeding ulcer are attributed to the use of NSAIDs, aspirin, or the twotaken in combination during the week prior to admission. (Faulkner G,Prichard P, Somerville K, et al. Aspirin and bleeding peptic ulcers inthe elderly. Br Med J. 1988; 297:1311-1313). A survey of TennesseeMedicaid patients who were hospitalized with GI complications showedthat patients who used NSAIDs had approximately a fourfold greater riskfor developing GI hemorrhage or peptic ulcer disease than patients nottaking NSAIDs. (Griffin M R, Piper J M, Daugherty J R, et al.Nonsteroidal anti-inflammatory drug use and increased risk for pepticulcer disease in elderly persons. Ann Intern Med. 1991; 114:257-263).Serious GI events, according to the FDA, occur in as many as 2% to 4% ofpatients per year who are taking continuous NSAID therapy for rheumatoidarthritis. The relative risk of gastric ulcer (4.725), duodenal ulcer(1.1 to 1.6), bleeding (3.8), perforation, and death are all increasedby NSAID use when such patients are compared to those who do not takethese products. In 1989, patients with rheumatoid arthritis hadapproximately 20,000 hospitalizations per year with an estimated cost of$ 10,000 per stay. (Fries J F, Miller S R, Spitz P W, et al. Toward anepidemiology of gastropathy associated with nonsteroidalanti-inflammatory drug use. J Gastroenterology. 1989; 96:647-655).

There is also a need for providing some of the NSAIDs in a water solubleform for injection. It is well known that high concentrations of alcoholand tromethamine used to form a salt in the current formulations ofKetorolac are toxic. At present there is no formulation that would allowthe NSAIDs to be in aqueous solution at the concentrations needed due topoor water solubility of the drug.

Beyond all these very evident practical difficulties lies the occurrenceof undesirable side effects already alluded to and observed, employingavailable oral dosage forms.

Several proposals to meet these various problems have been suggested inthe art, including both solid and liquid oral dosage forms. Anoverriding difficulty which has, however, remained is the inherentinsolubility of the NSAIDs in aqueous media, hence preventing the use ofa dosage form which can contain NSAIDs in sufficiently highconcentration to permit convenient use and yet meet the requiredcriteria in terms of bioavailability, e.g. enabling effective resorptionfrom the stomach or gut lumen and achievement of consistent andappropriately high blood/blood-serum levels.

The derivatives of the present invention with respect to NSAIDs overcomethe problems described hereinabove. More specifically, an embodiment ofthe present invention is directed to a derivative of the presentinvention of a NSAID which significantly enhances its solubility inaqueous solutions, thereby avoiding the need to utilize a carrier, suchas ethanol or castor oil when administered as a solution. Moreover, thederivatives of the present invention with respect to an NSAID do notexhibit the side effects of the prior art formulations. Further, thesederivatives of the present invention are almost completely devoid ofgastric irritability upon oral administration, thereby enhancingsignificantly the therapeutic index of the derivatives tested and theirefficacy.

Accordingly, in one aspect, the present invention is directed to aderivative of the present invention of NSAIDs, i.e., a L-Threoninecovalently bounded to the NSAID.

The preferred derivatives of the NSAIDs have the formula

or pharmaceutically acceptable salts thereof; wherein Y is either

in which either an amine group or the hydroxyl group of Threonine isreacted with the carboxylic acid group of the NSAIDs.

PCT application WO 2000/23419 of which Tom Jarvinen is an inventor,describes amino acid esters of NSAIDs, wherein the NSAIDs are attachedcovalently to amino acids via spacer groups, according the formula:

R—COO—R₁—O—R₂ where R₁ as defined therein is the spacer group, which maybe a saturated, unsaturated, straight chain, branched, or cyclicaklylene or alkylidene group of 1-8 carbon atoms, which can beoptionally substituted with 1-3 groups selected from halogen, hydroxyl,thiol, amino, mono- or dialkylamino, acylamino, carboxyl, alkylcarboxyl,acyl, aryl, aroyl, aralkyl, cyano, nitrol, alkoxy, alkenyloxy,alkylcarbonyloxy and arylcarbonyloxy derivatives, and R₂ as definedtherein is an aminoacyl residue of a synthetic or natural amino acid ofthe formula—C(═O)—R₃—NH₂and the preferred amino acids are naturally occurring ones such aslysine, proline, glycine and the like.

According to Jarvinen, these amino acid linked NSAIDs are suitable fortransdermal application only, and he gives examples of such derivativesmade with Naproxen.

There are several problems with these derivatives. For example, it isnot evident if these derivatives can be administered via oral route.Upon administration of any of these derivatives by any route, the bodyfluids will cause hydrolysis of the double esters leading to theformation of the NSAIDs, amino acid and finally the spacer group whichwill be a diol. All such diols produced in the body are highly toxic andhence the usefulness of this approach is limited.

However the present invention is directed towards L-Threonine esters ofNSAIDs, where the amino acids are attached to the NSAIDs directly viaester bond, thus avoiding toxicity of any spacer groups.

Contrary to the approach of Jarvinen and others, the current inventiononly releases the NSAIDs or the NSAID linked amino acid and once brokendown in the body, it releases, the amino acid, threonine, which is nottoxic. Hence the derivatives of the present invention are far superiorto their unesterified counterparts (i.e, intact NSAIDs) in terms oftoxicity, efficacy and therapeutic index.

The present invention is also directed to a pharmaceutical compositioncomprising a therapeutically effective amount of the various derivativesof the present invention of NSAIDs above and a pharmaceutical carriertherefor.

In another embodiment, the present invention is directed to a method oftreating a patient in need of NSAID therapy, which method comprisesadministering to said patient an effective amount of the derivative ofthe present invention of NSAIDs.

In a further embodiment, the present invention is directed to a methodof enhancing the solubility of NSAID in an aqueous solution comprisingreacting the carboxyl functionality of each of the NSAIDs withL-Threonine and isolating the products thereof.

In a still further embodiment, the present invention is directed to amethod of substantially and in a therapeutically efficacious manner,reducing or eliminating the gastric mucosal damage of NSAIDs whenadministered to a patient which comprises reacting the carboxylfunctionality of each of the NSAID molecule with either amine orhydroxyl function of L-Threonines to form either an amide or estercovalent bond respectively and isolating the product thereof andadministering said product to the patient.

A. Synthesis of Ibuprofen L-Threonine Derivaties

Overview:

The procedure for the synthesis of L-Threonine esters of Ibuprofen isoutlined in Synthetic Sequence section. The complete procedure andanalytical data is given in the Experimental Section. Again, thesesynthetic schemes are exemplary. In general, (±)-Ibuprofen (4-10 g, inbatches) was coupled with the N-benzyloxy/benzyl ester protectedL-Threonines (1 equivalent) with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1equivalent) in the presence of a catalytic amount of4-(N,N-dimethyamino)-pyridine (DMAP). Once the reactions were complete,any excess EDC was removed by extraction with water, DMAP was removed byextraction with dilute acid, and Ibuprofen was removed by extractionwith sodium bicarbonate. After drying over sodium sulfate, filtration,and concentration the crude protected L-Threonine esters of(±)-Ibuprofen were either used directly or purified by flashchromatography on silica gel to generate the protected esters in goodyield (85-95%). The column chromatography was generally not necessary ifa slight excess of Ibuprofen and coupling agent were used, and athorough extraction procedure was conducted. The protecting groups wereremoved by hydrogenation (25-35 psi H₂) in the presence of 10% palladiumon carbon and hydrochloric acid. Yields for the deprotection step rangedfrom 70-90%. The Threonine derivative was isolated as a solid.

After filtration and drying the hydrochloride salts of the serine andthreonine esters of (±)-Ibuprofen were purified by crystallization. Thehydrochloride salt of the L-hydroxyproline-Ibuprofen ester was a gelthat would not solidify/crystallize. In this case the hydrogenation wasrepeated without the use of acid and the neutral compound was purified.

Because the Ibuprofen started as a mixture of enantiomers, the finalproducts were delivered as a mixture of diastereomers. In the case ofthe threonine ester of Ibuprofen, washing with water, acetone oracetonitrile could readily separate the final diastereomeric salts. Theinsoluble isomer (SPI0016A) was determined to be the active isomer bycomparison with an authentic standard prepared from S-(+)-Ibuprofen.This synthesis repeated substituting L-serine and L-hydroxy-proline forL-Threonine. The serine and hydroxyproline esters of (±)-Ibuprofen couldnot be readily separated in this fashion.

Synthetic Sequence:

Synthesis of the L-serine, L-Threonine, and L-hydroxyproline esters of(±)-Ibuprofen: a) EDC, DMAP, CH₂Cl₂; b) HCl, 10% Pd/C, EtOH c) acetone,d) 10% Pd/C, EtOH.

Experimental Section:

The synthesis of SPI0015, SPI0016 and SPI0017 were conducted in two orthree batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Sigma-Aldrich, Acros, orBachem, except for solvents, which were purchased from either FisherScientific or Mallinkrodt.

1) Preparation of (±)-Ibuprofen-L-serine ester, hydrochloride (SPI0015).

(±)-Ibuprofen (5.04 g, 24.4 mmole), N-carbobenzyloxy-L-serine benzylester (8.11 g, 24.6 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 4.87g, 25.4 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.40 g, 3.27mmole) were dissolved in dichloromethane (150 mL) at room temperature,under an argon atmosphere. After stirring for 22 hours under an argonatmosphere at room temperature, water (100 mL) was added and the layerswere separated. The dichloromethane layer was washed again with water(100 mL) and dried for 1 hour over sodium sulfate (5 g). Afterfiltration and concentration under reduced pressure, the remaining oilwas purified by flash chromatography on silica gel (250 g), eluting withhexanes/ethyl acetate (3:1). The procedure generated the protectedL-serine-(±)-Ibuprofen ester (SPI001501) as a colorless solid (11.4 g,90% yield).

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-propionicacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.40-7.20 (m, 10H), 7.14-7.01 (m, 4H), 5.50(d, ½H, J=8.4 Hz), 5.29 (d, ½H, J=8.4 Hz), 5.11-5.02 (m, 2.5H), 4.90 (d,½H, J=12 Hz), 4.62 (m, 1H), 4.49-4.43 (m, 1H), 4.36-4.32 (m, 1H), 3.59(m, 1H), 2.39-2.35 (m, 2H), 1.78 (m, 1H), 1.42-1.39 (m, 3H), 0.85 (d,6H, J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=174.05, 169.19, 169.07, 155.68, 140.73,137.20, 136.12, 135.05, 134.91, 129.44, 128.67, 128.65, 128.60, 128.41,128.33, 128.30, 128.19, 127.19, 127.16, 67.75, 67.32, 64.51, 64.32,53.71, 45.16, 45.02, 30.35, 22.60, 18.27.

The protected Ibuprofen-L-serine ester (22.50 g, 43.4 mmole) wasdissolved in ethanol (200 mL) at room temperature and added to a Parrbottle that contained 10% palladium on carbon (3.86 g, 50% wet) under anitrogen atmosphere. Hydrochloric acid (10 mL 37% HCl in 30 mL water)was added and the nitrogen atmosphere was replaced with hydrogen gas (25psi). After 4 hours of shaking, the palladium catalyst was removed byfiltration through celite. The ethanol/water was removed under reducedpressure. The remaining white solids were washed with water (25 mL),acetone (20 mL) and dried under high vacuum (4 hours at 88° C.). Theexperiment produced (±)-Ibuprofen-L-serine ester, hydrochloride SPI0015(11.3 g, 80% yield) as a colorless solid.

2(S)-Amino-3-[2(R,S)-(4-isobutylphenyl)-propionyloxy]-propionic acid,hydrochloride; ((R,S)-Ibuprofen-L-Serine ester, hydrochloride)

¹H NMR (300 MHz, DMSO): δ=8.92 (br s, 3H), 7.22 (t, 2H, J=7.5 Hz), 7.10(d, 2H, J=7.5 Hz), 4.56 (m, 1H), 4.37-4.20 (m, 2H), 3.83 (q, 1H, J=6.9Hz), 2.41 (d, 2H, J=6.9 Hz), 1.80 (m, 1H), 1.41 (d, 3H, J=6.9 Hz), 0.85(d, 6H, J=6.9 Hz).

¹³C NMR (75 MHz, DMSO): δ=173.36, 173.32, 168.08, 168.04, 139.70,128.96, 129.92, 127.20, 127.05, 62.47, 51.59, 51.49, 44.28, 44.00,43.90, 29.68, 22.28, 18.70, 18.42.

HPLC Analysis:

99.13% purity; rt=3.133 min; Luna C18 5u column (sn 167917-13); 4.6×250mm; 254 nm; 50% ACN/50% TFA buffer (0.1%); 35 C; 20 ul inj.; 1 ml/min; 1mg/mL sample size; sample dissolved in mobile phase.

CHN Analysis:

calc.: C, 58.27; H, 7.33; N, 4.25; found: C, 58.44; H, 7.46; N, 4.25.

Melting point: 169.5-170.5° C.

2a) Preparation and Separation of (±)-Ibuprofen-L-Threonine ester,hydrochloride (SPI0016A and SPI0016B).

(±)-Ibuprofen (4.15 g, 20.11 mmole), N-carbobenzyloxy-L-Threonine benzylester (6.90 g, 20.11 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 3.95g, 20.6 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.25 g, 2.0mmole) were dissolved in dichloromethane (50 mL) at room temperature,under an argon atmosphere. After stirring for 19 hours, thedichloromethane layer was washed with water (50 mL), 5% hydrochloricacid (2×25 mL), water (25 mL), saturated sodium bicarbonate (2×25 mL),and water (50 mL). After drying for one hour over sodium sulfate (5 g),filtration, and concentration under reduced pressure, the remaining oilwas used without further purification. The procedure generated theprotected L-Threonine-(±)-Ibuprofen ester (SPI001601) as a light yellowoil (10.2 g, 95.3% yield), which solidified on standing.

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyricacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.40-7.15 (m, 10H), 7.14-7.01 (m, 4H),5.48-5.25 (m, 2H), 5.11-5.01 (m, 3H), 4.90 (d, ½H, J=12 Hz), 4.68 (d,½H, J=12 Hz), 4.48 (m, 1H), 3.60-3.48 (m, 1H), 2.39(m, 2H), 1.79 (m,1H), 1.42-1.35 (m, 3H), 1.27 (d, 1.5H, J=6.6 Hz), 1.17 (d, 1.5H, J=6.6Hz), 0.85 (m, 6H).

¹³C NMR (75 MHz, CDCl₃): δ=173.32, 169.70, 169.30, 156.55, 140.75,137.38, 137.22, 136.14, 135.07, 134.99, 129.45, 129.41, 128.65, 128.39,128.22, 127.21, 127.14, 70.97, 70.70, 67.81, 67.66, 67.53, 57.83, 45.19,30.39, 22.61, 18.57, 18.30, 17.18, 16.87.

The protected Ibuprofen-L-Threonine ester (10.15 g, 19.0 mmole) wasdissolved in warm ethanol (150 mL) and added to a Parr bottle thatcontained 10% palladium on carbon (3.4 g, 50% wet) under a nitrogenatmosphere. Hydrochloric acid (6 mL 37% HCl in 20 mL water) was addedand the nitrogen atmosphere was replaced with hydrogen gas (30 psi).After 3 hours of shaking, the palladium catalyst was removed byfiltration through celite (30 g). The ethanol/water was removed underreduced pressure. The experiment produced (±)-Ibuprofen-L-Threonineester, hydrochloride (SPI0016A and SPI0016B, 6.4 g, 97% crude yield) asa colorless solid. The crude mixture of diastereomers was stirred inacetone (200 mL) for 2 hours at room temperature under an argonatmosphere. After 2 hours the solids (2.84 g, SPI0016A) were filtered.The filtrate (SPI0016B, 3.0 g) was concentrated under reduced pressure.

Purification of SPI0016A (Active Isomer):

After 3 batches of the S-Ibuprofen-L-Threonine ester (SPI0016A) had beencompleted, the batches were combined (8.78 g total) and crystallizedthree times from deionited ultra filtered water (“DIUF”) (100 mL). Eachtime a small amount of zwitterion was generated. In order to regeneratethe salt, the solid generated (from each crystallization) was dissolvedin 1% hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mLethanol). The ethanol solution was then concentrated under reducedpressure at room temperature. After the third crystallization andregeneration procedure, the salt (5.6 g) was stirred in acetonitrile(100 mL) for 44 hours at room temperature, under an argon atmosphere.The salt was then filtered and dried under high vacuum at 50-55 untilthe weight was constant (5.5 g).

2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(S-Ibuprofen-L-Threonine ester, Hydrochloride, Active Isomer)

¹H NMR (300 MHz, DMSO): δ=8.76 (br s, 3H), 7.19 (d, 2H, J=8.1 Hz), 7.11(d, 2H, J=8.1 Hz), 5.28 (dq, 1H, J=6.3, 3.6 Hz), 4.14 (q, 1H, J=3.6 Hz),3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H, J=7.2 Hz), 1.80 (m, 1H), 1.37 (d,3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3 Hz), 0.85 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=172.66, 168.24, 139.68, 137.24, 128.95,126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

CHN Analysis:

calc.: C, 59.38; H, 7.62; N, 4.07; found: C, 59.17; H, 7.63; N, 4.04.

HPLC Analysis:

98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical rotation: +24.5°

Melting Point: 189-190° C.

2) Purification of SPI0016B (Inactive Isomer):

After 3 batches of the R-Ibuprofen-L-Threonine ester (SPI0016B) had beencompleted, the batches were combined (9.02 g total) and crystallizedfrom DIUF water (50 mL). A small amount of zwitterion was generatedduring the crystallization. In order to regenerate the salt, the solidgenerated was dissolved in 1% hydrochloric acid in ethanol (3 mL 37%hydrochloric acid in 100 mL ethanol). The ethanol solution was thenconcentrated under reduced pressure at room temperature. The remainingsalt (5.93 g) was crystallized three times from hot toluene (100 mL)with the addition of a small amount on acetone (1 mL). The salt was thenfiltered and dried under high vacuum at room temperature until theweight was constant (5.1 g).

2(S)-Amino-3(R)-[2(R)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(R-Ibuprofen-L-Threonine ester, Hydrochloride, Inactive Isomer)

¹H NMR (300 MHz, DMSO): δ=8.82 (br s, 3H), 7.23 (d, 2H, J=7.8 Hz), 7.10(d, 2H, J=7.8 Hz), 5.27 (m, 1H), 4.18 (m, 1H), 3.80 (q, 1H, J=7.2 Hz),2.41 (d, 2H, J=7.2 Hz), 1.81 (m, 1H), 1.41 (d, 3H, J=6.9 Hz), 1.34 (d,3H, J=6.3 Hz), 0.85 (d, 6H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=72.56, 168.08, 139.64, 136.98, 128.84, 127.14,68.8, 55.29, 44.28, 29.69, 22.28, 18.24, 16.41.

CHN Analysis:

calc.: C, 59.38; H, 7.62; N, 4.07; found: C, 59.30; H, 7.60; N, 4.05.

HPLC Analysis:

98.43% purity; r.t.=6.19 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical Rotation: +10.4°

Melting Point: 176-177° C.

2b) Preparation of the S-(+)-Ibuprofen-L-Threonine ester, HydrochlorideStandard (SPI0016S)

S-(+)-Ibuprofen (2.0 g, 9.69 mmole), N-carbobenzyloxy-L-Threonine benzylester (3.25 g, 9.91 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1.90g, 9.91 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12 g, 1.0mmole) were dissolved in dichloromethane (25 mL) at room temperature,under an argon atmosphere. After stirring for 4 hours, thedichloromethane layer was washed with water (25 mL), 5% hydrochloricacid (25 mL), saturated sodium bicarbonate (2×25 mL), and water (25 mL).After drying for one hour over sodium sulfate (5 g), filtration, andconcentration under reduced pressure, the remaining oil was used withoutfurther purification. The procedure generated the protectedS-(+)-lbuprofen-L-Threonine ester (SPI001601S) as a light yellow oil(5.01 g, 98% yield), which solidified on standing.

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyricacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.35-7.23 (m, 10H), 7.10 (d, 2H, J=7.8 Hz),7.05 (d, 2H, J=7.8 Hz), 5.48-5.25 (m, 2H), 5.17-5.01 (m, 4H), 4.50 (dd,1H, J=9.6, 1.8 Hz), 3.50 (q, 1H, J=7.2 Hz), 2.40 (d, 2H, J=7.2 Hz), 1.80(m, 1H), 1.37 (d, 3H, J=7.2 Hz), 1.17 (d, 3H, J=6.3 Hz), 0.86 (d, 6H,J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=173.29, 169.69, 156.51, 140.68, 137.21,136.08, 135.06, 129.40, 128.70, 128.66, 128.57, 128.38, 128.24, 127.14,70.70, 67.80, 67.53, 57.87, 45.19, 45.11, 30.39, 22.61, 18.57, 16.87.

The protected S-(+)-Ibuprofen-L-Threonine ester (5.0 g, 9.40 mmole) wasdissolved in warm ethanol (100 mL) and added to a Parr bottle thatcontained 10% palladium on carbon (1.0 g, 50% wet) under a nitrogenatmosphere. Hydrochloric acid (1 mL 37% HCl in 10 mL water) was addedand the nitrogen atmosphere was replaced with hydrogen gas (32 psi).After 2 hours of shaking, the palladium catalyst was removed byfiltration through celite (30 g). The ethanol/water was removed underreduced pressure. The experiment produced S-(+)-Ibuprofen-L-Threonineester, hydrochloride (SPI0016S, 2.8 g, 85% crude yield) as a colorlesssolid. The salt was stirred in acetone (50 mL) for 3 hours at roomtemperature under an argon atmosphere. After 3 hours the solids (2.24 g,69% yield) were filtered and dried under high vacuum at roomtemperature, until the weight was constant.

2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(S-Ibuprofen-L-Threonine ester, Hydrochloride, Active Isomer)

¹H NMR (300 MHz, DMSO): δ=8.76 (br s, 3H), 7.19 (d, 2H, J=8.1 Hz), 7.11(d, 2H, J=8.1 Hz), 5.28 (dq, 1H, J=6.3, 3.6 Hz), 4.14 (q, 1H, J=3.6 Hz),3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H, J=7.2 Hz), 1.80 (m, 1H), 1.37 (d,3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3 Hz), 0.85 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=172.66, 168.24, 139.68, 137.24, 128.95,126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

HPLC Analysis:

98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical rotation: +26.5°

Melting Point: 189-190° C.

3) Preparation of the (±)-Ibuprofen-L-hydroxyproline ester (SPI0017).

(±)-Ibuprofen (5.10 g, 24.7 mmole), N-carbobenzyloxy-L-hydroxyprolinebenzyl ester (8.80 g, 24.7 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 5.10g, 26.0 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.30 g, 2.40mmole) were dissolved in dichloromethane (100 mL) at room temperature,under an argon atmosphere. After stirring for 24 hours under an argonatmosphere at room temperature, water (100 mL) was added and the layerswere separated. The dichloromethane layer was washed again with water(100 mL), 5% sodium bicarbonate (2×50 mL) and dried for 1 hour oversodium sulfate (5 g). After filtration and concentration under reducedpressure, the remaining oil was used without further purification. Theprocedure generated the protected (±)-Ibuprofen-L-hydroxyproline ester(SPI001701) as a light yellow oil (11.5 g, 85% yield).

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid; ((R,S)-Ibuprofen-L-hydroxyproline ester)

¹H NMR (300 MHz, CDCl₃): δ=7.33-7.02 (m, 14H), 5.25-4.95 (m, 5H),4.51-4.19 (m, 1H), 3.75-3.50 (m, 3H), 2.40 (d, 2H, J=6.9 Hz), 2.15 (m,1H), 1.81 (m, 1H), 1.44 (d, 3H, J=7.0 Hz), 0.87 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=173.99, 171.93, 171.72, 154.68, 154.15,140.70, 137.23, 137.04, 136.23, 135.44, 135.23, 129.41, 128.59, 128.47,128.35, 128.19, 128.08, 127.89, 127.02, 72.86, 72.16, 67.40, 67.18,67.09, 58.12, 57.83, 52.66, 52.49, 52.13, 45.15, 36.63, 35.67, 32.07,30.33, 29.23, 22.90, 22.58, 18.36.

The protected Ibuprofen-L-hydroxyproline ester (11.40 g, 43.4 mmole) wasdissolved in ethanol (150 mL) at room temperature and added to a Parrbottle that contained 10% palladium on carbon (2.73 g, 50% wet) under anitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogengas (34 psi). After 5 hours of shaking, the palladium catalyst wasremoved by filtration through celite. The ethanol was removed underreduced pressure. The remaining white solids (6.60 g) were washed withDIUF water (50 mL), diethyl ether (50 mL) and dried under high vacuumuntil the weight was constant. The experiment produced(±)-Ibuprofen-L-hydroxyproline ester SPI0017 (5.64 g, 84% yield) as acolorless solid.

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid; ((R,S)-Ibuprofen-L-hydroxyproline ester)

¹H NMR (300 MHz, CDCl₃): δ=7.22 (d, 2H, J=7.2 Hz), 7.09 (d, 2H, J=7.2Hz), 5.27 (m, 1H), 4.40 (t, 0.5H, J=7 Hz), 4.24 (t, 0.5H, J=9 Hz), 3.75(m, 1H), 3.61(m, 1H), 3.28 (d, 0.5H, J=13 Hz), 3.15 (d, 0.5H, J=13 Hz),2.42-2.10 (m, 4H), 1.78 (m, 1H), 1.40 (br t, 3H, J=6 Hz), 0.82 (d, 6H,J=6 Hz). (mixture of diastereomers)

¹³C NMR (75 MHz, CDCl₃): δ=173.28, 173.23, 168.98, 139.88, 137.33,137.23, 129.12, 127.26, 127.17, 72.58, 57.60, 57.50, 50.24, 50.12,44.34, 44.15, 34.31, 34.16, 29.77, 22.34, 18.43, 18.23. (mixture ofdiastereomers)

HPLC Analysis:

100% purity; r.t.=5.35, 5.22 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min;32.3 C, Luna C18, serial # 188255-37; 20 ul inj.

CHN Analysis:

calc.: C, 67.69; H, 7.89; N, 4.39; found: C, 67.47; H, 7.87; N, 4.30.

Melting Point: 198-199° C.

Overview Ketoprofen S(+) Threonine Ester Synthesis:

The procedure for the synthesis of the L-Threonine esters of Ketoprofenis outlined in the Synthetic Sequence section. The complete procedureand analytical data is given in the Experimental Section. In general,(±)-Ketoprofen (5 g) was coupled with N-boc-L-Threonine t-butyl ester (1equivalent) with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 1 equivalent) in the presence of a catalytic amountof 4-(N,N-dimethyamino)-pyridine (DMAP). Once the reaction was complete,any excess EDC was removed by extraction with water, DMAP was removed byextraction with dilute acid, and Ketoprofen was removed by extractionwith sodium bicarbonate. After drying over sodium sulfate, filtration,and concentration the crude protected L-Threonine-(±)-Ketoprofen waspurified by flash chromatography on silica gel to generate the protectedL-Threonine ester in good yield (98%). The protecting groups wereremoved by treatment with 2M hydrochloric acid in diethyl ether tocleave the boc group, followed by treatment with trifluoroacetic acid toremove the t-butyl ester. After drying, the mixture ofL-Threonine-R,S(±)-Ketoprofen esters was separated by crystallizationfrom acetonitrile. The hydrochloride salt of theL-Threonine-S(+)-Ketoprofen ester preferentially precipitated fromacetonitrile. A sample of an optically pure standard was preparedstarting with S(+)-ketoprofen for comparison. After drying and analysis,a sample of L-Threonine-S(+)-Ketoprofen ester, hydrochloride (1.75 g)was separated from the mixture.

Synthetic Sequence:

Synthesis of the L-Threonine esters of (±)-Ketoprofen: a) EDC, DMAP,CH₂Cl₂; b) HCl (2M); c) TFA; d) ACN (crystallization).

Experimental Section:

The synthesis of SPI0018A was conducted in a single batch. Reagentsmentioned in the experimental section were purchased at the highestobtainable purity from Sigma-Aldrich, Acros, or Bachem, except forsolvents, which were purchased from either Fisher Scientific orMallinkrodt.

Preparation and Separation of S(+)-Ketoprofen-L-Threonine ester,Hydrochloride (SPI0018A).

(±)-Ketoprofen (5.32 g, 20.92 mmol), N-t-butylcarbonyl-L-Threoninet-butyl ester (Boc-Thr-OtBu (5.17 g, 18.72 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 4.0g, 20.9 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.22 g) weredissolved in dichloromethane (50 mL) at room temperature, under an argonatmosphere. After stirring for 5 hours, the dichloromethane layer waswashed with water (50 mL), 5% hydrochloric acid (2×25 mL), water (25mL), saturated sodium bicarbonate (2×25 mL), and water (50 mL). Afterdrying for one hour over sodium sulfate (5 g), filtration, andconcentration under reduced pressure, the remaining oil (10.3 g) waspurified by column chromatography on silica gel (150 g), eluting withhexanes/ethyl acetate (2:1). After combining the product containingfractions, concentration and drying under high vacuum, the proceduregenerated the protected L-Threonine-(±)-Ketoprofen ester (SPI001801) asa clear oil (9.42 g, 98% yield).

3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-butyricacid tert-butyl ester: (Mix of Diastereomers)

¹H NMR (300 MHz, CDCl₃): δ=7.83-7.42 (m, 9H), 5.43 (dd, 1H, J=13.2, 6.9Hz), 5.10 (dd, 1H, J=20.7, 9.3), 4.29 (t, 1H, J=11.7 Hz), 3.75 (q, 1H,J=7.2 Hz), 1.50-1.42 (m, 19.5H), 1.30-1.18 (m, 4.5H).

¹³C NMR (75 MHz, CDCl₃): δ=. 196.18, 172.62, 172.55, 168.85, 168.58,155.81, 140.33, 140.23, 137.86, 137.39, 132.46, 132.42, 131.54, 131.38,130.00, 129.31, 129.13, 129.02, 128.54, 128.27, 82.50, 82.37, 80.05,71.38, 71.22, 57.59, 57.52, 45.46, 45.31, 28.40, 27.98, 27.84, 18.54,18.48, 17.19, 16.84.

The protected (R,S)-Ketoprofen-L-Threonine ester (9.42 g, 18.41 mmol)was dissolved in dichloromethane (25 mL) under an argon atmosphere, atroom temperature. Anhydrous hydrochloric acid in diethyl ether (2M, 25mL) was added to the solution and the mixture was allowed to stir for 17hours at room temperature. The mixture was concentrated under reducedpressure. The remaining foam (8.2 g) was dissolved in a mixture ofdichloromethane (10 mL) and trifluoroacetic acid (20 mL). After stirringat room temperature for 6.5 hours the solution was concentrated underreduced pressure. Toluene (25 mL) was added to the remaining oil and themixture was concentrated a second time. A mixture of ethanol (20 mL) andanhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added andthe solution was concentrated a third time. After drying under highvacuum for 2 hours at room temperature, the experiment produced(±)-Ketoprofen-L-Threonine ester, hydrochloride (mix of diastereomers,7.11 g, 98% crude yield) as an off-white solid. The crude mixture ofdiastereomers (7.0 g) was crystallized 3 times from acetonitrile (200mL). After the third crystallization, the remaining white solid wasdried under high vacuum at 50° C. until the weight was constant (4hours). The experiment produced L-Threonine-S(+)-Ketoprofen ester,hydrochloride SPI0018A (2.2 g, 30% yield from SPI001801).

2(S)-Amino-3(R)-[2(S)-(3-benzoyl-phenyl)-propionyloxy]-butyric acid,hydrochloride (L-Threonine-S(+)-Ketoprofen ester, Hydrochloride)

¹H NMR (300 MHz, DMSO): δ=14.08 (br s, 1H), 8.72 (br s, 3H), 7.74-7.51(m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m, 1H), 3.97 (q, 1H, J=6.3 Hz),1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=195.34, 172.26, 168.21, 140.42, 137.05,136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31,44.00, 18.44, 16.45.

CHN Analysis:

calc.: C, 61.30; H, 5.66; N, 3.57; found: C, 61.02; H, 5.58; N, 3.58.

HPLC Analysis:

98.28% purity; r.t.=25.14 min.; 55% DIUF water (0.1% TFA)/45% methanol;1 mL/min; 36.4 C; Luna C18, 5u column (serial # 211739-42), 4.6×250 mm;20 ul injection.

Optical rotation: +27.0° (20 C, 174.4 mg/10 mL ethanol, 589 nm); MeltingPoint: 166-167° C.

Preparation of the S-(+)-Ketoprofen-L-Threonine ester, HydrochlorideStandard.

(+)-Ketoprofen (1.87 g, 7.74 mmol), N-t-butylcarbonyl-L-Threoninet-butyl ester (Boc-Thr-OtBu, 2.25 g, 8.14 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1.65g, 8.60 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.1 g) weredissolved in dichloromethane (25 mL) at room temperature, under an argonatmosphere. After stirring for 4 hours, the dichloromethane layer waswashed with water (25 mL). After drying for one hour over sodium sulfate(5 g), filtration, and concentration under reduced pressure, theremaining oil was used without purification. The procedure generated theprotected L-Threonine-(+)-Ketoprofen ester as a clear oil (4.01 g, ˜100%yield).

¹H NMR (300 MHz, CDCl₃): δ=7.81-7.42 (m, 9H), 5.43 (m, 1H), 5.10 (d, 1H,J=9.3), 4.29 (d, 1H, J=9.6 Hz), 3.75 (q, 1H, J=7.2 Hz), 1.50-1.42 (m,21H), 1.18 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=196.4, 172.79, 168.99, 155.94, 140.44,137.99, 137.51, 132.59, 131.50, 130.13, 129.31, 129.25, 129.15, 128.66,128.40, 82.68, 80.24, 71.37, 57.71, 45.43, 28.53, 28.10, 18.99, 16.96.

The protected (S)-Ketoprofen-L-Threonine ester (3.92 g, 7.66 mmol) wasdissolved in anhydrous hydrochloric acid in diethyl ether (2M, 50 mL)and stirred for 17 hours at room temperature. The mixture wasconcentrated under reduced pressure. The remaining foam (3.4 g) wasdissolved in a mixture of dichloromethane (20 mL) and trifluoroaceticacid (20 mL). After stirring at room temperature for 6.5 hours thesolution was concentrated under reduced pressure. Toluene (25 mL) wasadded to the remaining oil and the mixture was concentrated a secondtime. A mixture of ethanol (20 mL) and anhydrous hydrochloric acid indiethyl ether (2M, 20 mL) was added and the solution was concentrated athird time. After drying under high vacuum for 2 hours at roomtemperature, the experiment produced S(+)-Ketoprofen-L-Threonine ester,hydrochloride (3.05 g crude) as an off-white solid. The crude materialwas stirred with acetone (50 mL) for 2 hours at room temperature underan argon atmosphere. The remaining white solid was filtered and driedunder high vacuum at 50° C. until the weight was constant (4 hours). Theexperiment produced L-Threonine-S(+)-Ketoprofen ester, hydrochloride(2.04 g, 67% yield).

¹H NMR (300 MHz, DMSO): δ=14.08 (br s, 1H), 8.72 (br s, 3H), 7.74-7.51(m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m, 1H), 3.97 (q, 1H, J=6.3 Hz),1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=195.34, 172.26, 168.21, 140.42, 137.05,136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31,44.00, 18.44, 16.45.

HPLC Analysis:

99.43% purity; r.t.=25.14 min.; 55% DIUF water (0.1% TFA)/45% methanol;1 mL/min; 36.4 C; Luna C18, 5u column (serial # 211739-42), 4.6×250 mm;20 ul injection.

Optical rotation: +27.1° (20 C, 177.8 mg/10 mL ethanol, 589 nm); MeltingPoint: 166-167° C.

Preparation of the (±)Ketoprofen-L-serine ester, hydrochloride

(±)-Ketoprofen (7.30 g, 28.7 mmol), N-t-butylcarbonyl-L-serine t-butylester (Boc-Ser-OtBu, (7.50 g, 28.7 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 5.5g, 28.7 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12 g) weredissolved in dichloromethane (50 mL) at room temperature, under an argonatmosphere. After stirring for 3 hours, the dichloromethane layer waswashed with water (50 mL), 5% hydrochloric acid (2×25 mL), water (25mL), saturated sodium bicarbonate (2×25 mL), and water (50 mL). Afterdrying for one hour over sodium sulfate (5 g), filtration, andconcentration under reduced pressure, the remaining foam was usedwithout purification. The procedure generated the protectedL-serine-(±)-Ketoprofen ester as a clear foam (13.72 g, 96% yield).

3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-propionicacid tert-butyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.77-7.38 (m, 9H), 5.29 (d, ½H, J=6.9 Hz),5.13 (d, ½H, J=6.9 Hz), 4.44-4.30 (m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50(d, 3H, J=7 Hz), 1.39 (m, 18H).

¹³C NMR (75 MHz, CDCl₃): δ=. 196.13, 173.37, 168.37, 154.99, 140.40,137.89, 132.50, 131.44, 130.01, 129.25, 129.14, 128.53, 128.29, 82.72,80.03, 65.22, 64.91, 53.62, 53.40, 45.29, 28.41, 28.02, 27.91, 18.59,18.50.

The protected (R,S)-Ketoprofen-L-serine ester (13.6 g, 127.31 mmol) wasdissolved in anhydrous hydrochloric acid in diethyl ether (2M, 100 mL)under an argon atmosphere, at room temperature. The mixture was allowedto stir for 23 hours at room temperature when dichloromethane was added(100 mL). After 48 hours, the mixture was concentrated under reducedpressure. The remaining light yellow foam (9.0 g) was dissolved in amixture of dichloromethane (200 mL) and DIUF water (50 mL). After mixingat room temperature, the layers were separated. The dichloromethanelayer was acidified with 2N hydrochloric acid in ether (5 mL) dried oversodium sulfate (10 g) filtered and concentrated under reduced pressure.The remaining foam (6.4 g) was stirred with dichloromethane (40 mL) for30 minutes at room temperature under an argon atmosphere. Diethyl etherwas added (20 mL) and the mixture was allowed to stir for 2 hours atroom temperature. After 2 hours the solids were filtered and dried underhigh vacuum at room temperature until a constant weight was obtained.The experiment produced L-serine-R,S(±)-Ketoprofen ester, hydrochloride(2.5 g, 22% yield).

2(S)-Amino-3-[2(R,S)-(3-benzoyl-phenyl)-propionyloxy]-propionic acid,hydrochloride

H NMR (300 MHz, DMSO): δ=8.79 (br s, 3H), 8.72 (br s, 3H), 7.76-7.54 (m,9H), 4.57 (m, 1H), 4.42-4.28 (m, 2H), 4.01 (m, 1H), 1.46 (d, 3H, J=6Hz).

¹³C NMR (75 MHz, DMSO): δ=195.33, 172.92, 168.01, 167.96, 140.50,140.39, 136.97 (d), 136.75, 132.66, 131.93 (d), 129.55, 128.65 (d),128.49 (d), 62.18, 51.35 (d), 44.07, 18.62, 18.41.

HPLC Analysis:

98.99% purity; r.t.=9.205 min. (broad peak); 55% DIUF water (0.1%TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5u column (serial #211739-42), 4.6×250 mm; 20 ul injection.

CHN Analysis:

calc.: C, 60.40; H, 5.34; N, 3.71; found: C, 60.15; H, 5.32; N, 3.72.

Melting Point: 116-120° C. (uncorrected)

Preparation of the (±)Ketoprofen-L-hydroxyproline ester, Hydrochloride

(±)-Ketoprofen (6.70 g, 26.3 mmol),N-t-butylcarbonyl-trans-L-hydroxyproline-t-butyl ester (Boc-Hyp-OtBu,7.40 g, 25.7 mmol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 5.25 g, 27.3 mmol), and4-(N,N-dimethylamino)-pyridine (DMAP, 0.10 g) were dissolved indichloromethane (50 mL) at room temperature, under an argon atmosphere.After stirring for 3.5 hours, the dichloromethane layer was washed withwater (50 mL), 5% hydrochloric acid (2×25 mL), water (25 mL), saturatedsodium bicarbonate (2×25 mL), and water (50 mL). After drying for onehour over sodium sulfate (5 g), filtration, and concentration underreduced pressure, the remaining light green oil (13.30 g) was usedpurified by column chromatography on silica gel (120 g), eluting withheptane/ethyl acetate (2:1). After combining the product containingfractions, concentration under reduced pressure and drying under highvacuum, the procedure generated the protectedL-hydroxyproline-(±)-Ketoprofen ester as a clear oil (5.50 g, 41%yield).

4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-1,2(S)-dicarboxylicacid di-tert-butyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.77-7.38 (m, 9H), 5.29 (d, ½H, J=6.9 Hz),5.13 (d, ½H, J=6.9 Hz), 4.44-4.30 (m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50(d, 3H, J=7 Hz), 1.39 (m, 18H).

¹³C NMR (75 MHz, CDCl₃): δ=196.25, 173.43 (d), 171.46 (d), 153.66 (d),140.35, 138.00, 137.47, 132.55, 131.38, 130.05, 129.13, 128.67, 128.30,81.55, 80.37 (d), 73.31, 72.48, 58.56, 51.86 (d), 45.43, 36.68 (d),28.49, 28.18, 18.60.

The protected (R,S)-Ketoprofen-L-hydroxyproline ester (3.30 g, 6.31mmol) was dissolved in anhydrous hydrochloric acid in diethyl ether (2M,20 mL) under an argon atmosphere, at room temperature. After 72 hours,the mixture was concentrated under reduced pressure. The remaining lightyellow foam (2.6 g) was dissolved in a mixture of dichloromethane (50mL) and DIUF water (10 mL). After mixing at room temperature, the layerswere separated. The dichloromethane layer was acidified with 2Nhydrochloric acid in ether (5 mL) dried over sodium sulfate (5 g)filtered and concentrated under reduced pressure. The remaining foam (2g) was stirred with diethyl ether (20 mL) for 30 minutes at roomtemperature under an argon atmosphere. The solids were filtered anddried under high vacuum at room temperature until a constant weight wasobtained. The experiment produced L-hydroxyproline-R,S(±)-Ketoprofenester, hydrochloride (1.2 g, 48% yield).

4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid, Hydrochloride

H NMR (300 MHz, DMSO): δ=10.25 (br s, 2H), 7.73-7.53 (m, 9H), 5.29 (brm, 1H), 4.38 (t, ½H, J=8.1 Hx), 4.26 (t, ½H, J=9 Hz), 3.95 (m, 1H), 3.60(m, 1H), 3.28 (d, ½H, J=13 Hz), 3.16 (d, ½H, J=12 Hz), 2.37-2.20 (m,2H), 1.45 (m, 3H).

¹³C NMR (75 MHz, DMSO): δ=195.38, 172.78, 172.73, 169.16, 140.50,140.41, 137.08, 136.77, 132.67, 132.01, 131.89, 129.52, 128.78, 128.50,128.50, 72.87 (d), 57.60, 57.52, 50.16 (d), 44.30, 44.20, 34.26, 34.15,18.43, 18.25.

HPLC Analysis:

99.99% purity; r.t.=7.842 and 7.689 min. (broad double peak); 55% DIUFwater (0.1% TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5u column(serial # 211739-42), 4.6×250 mm; 20 ul injection.

CHN Analysis:

calc.: C, 62.45; H, 5.49; N, 3.47; found: C, 61.78; H, 5.56; N, 3.62.

Melting Point: 170-173° C. (uncorrected).

These synthetic procedures demonstrate that L-threonine is capable ofreadily separating Ketoprofen into its respective enantiomeric esters,while other the hydroxyl amino acids such as Hydroxyproline or serinedid not do so readily.

Synthesis of Keotorlac-L-threonine ester and Human Trials:

Overview:

The procedure for the synthesis of the L-threonine ester of Ketorolac isoutlined in Synthetic Sequence section. The complete procedure andanalytical data is given in the Experimental Section. In general,(±)-Ketorolac was extracted from the tromethamine salt (10 g) andcoupled with N-boc-L-threonine t-butyl ester (1 equivalent) with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDCI) inthe presence of a catalytic amount of 4-(N,N-dimethylamino)-pyridine(DMAP). The crude protected L-threonine-(±)-Ketorolac ester was purifiedby flash chromatography. The protecting groups were removed by treatmentwith trifluoroacetic acid. The mixture of L-threonine-R,S(±)-Ketorolacester salts was separated by crystallization from acetonitrile/acetone.A sample of S(−)-Ketorolac L-threonine ester, hydrochloride (2.1 g)separated from the mixture was shipped to Signature for testing.

Synthesis of the L-threonine esters of (±)-Ketorolac: a) AcOH/H₂O,CH₂Cl₂; b) EDC, DMAP, CH₂Cl₂; c) TFA; d) HCl, ethanol; e) ACN-acetone(crystallization).

Experimental Section:

The synthesis of SPI0031A was conducted in a single batch. The procedurewas later repeated to ensure reproducibility. Reagents mentioned in theexperimental section were purchased at the highest obtainable purityfrom Cayman Chemical, Sigma-Aldrich, Acros, or Bachem, except forsolvents, which were purchased from either Fisher Scientific orMallinkrodt.

Preparation and Separation of S(−)-Ketorolac L-threonine ester,Hydrochloride (SPI0031A).

(±)-Ketrolac tromethamine salt (10 g, Cayman Chemical) was dissolved inwater (100 mL), acetic acid (20 mL), and dichloromethane (50 mL). Aftermixing for ten minutes, the layers were separated and the water fractionwas extracted two additional times with dichloromethane (50 mL). Thedichloromethane fractions were combined, dried over sodium sulfate,filtered, concentrated, and dried under high vacuum at room temperatureuntil a constant weight was obtained. The procedure generated(±)-Ketrolac (6.78 g, 100% yield) as an off-white solid.

¹H NMR (300 MHz, CDCl₃): δ 9.62 (1H, br s), 7.80 (2H, d, J=6.9 Hz),7.55-7.42 (3H, m), 6.84 (1H, d, J=4.0 Hz), 6.15 (1H, d, J=4.0 Hz),4.62-4.41 (2H, m), 4.10 (1H, dd, J=8.4, 5.7 Hz), 2.97-2.75 (2H, m).

¹³C NMR (75 MHz, CDCl₃): δ=185.25, 176.69, 142.04, 139.05, 131.61,129.02, 128.26, 127.31, 125.43, 103.63, 47.77, 42.64, 31.20.

(±)-Ketrolac (6.80 g, 26.6 mmol), N-tert-butylcarbonyl-L-threoninetert-butyl ester (Boc-Thr-OtBu, 7.33 g, 26.6 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDCI, 5.50g, 28.6 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.10 g) weredissolved in dichloromethane (75 mL) at room temperature, under an argonatmosphere. After stirring for 6 hours, the dichloromethane solution waswashed with water (50 mL), saturated sodium bicarbonate 50 mL), andwater (50 mL). After drying the dichloromethane solution for one hourover sodium sulfate (10 g), filtration, and concentration under reducedpressure, the remaining brown oil (14.55 g) was purified by columnchromatography on silica gel (250 g), eluting with heptane/ethyl acetate(1:1). After combining the product containing fractions, concentrationand drying under high vacuum, the procedure generated the protectedL-threonine-(±)-Ketorolac ester (SPI003101) as light brown solid foam(13.53 g, 99.2% yield).

¹H NMR (300 MHz, CDCl₃): δ=7.80 (2H, m), 7.55-7.42 (3H, m), 6.81 (1H,m), 6.08 (1H,m), 5.47 (1H, m), 5.17 (1H, m), 4.60-4.34 (3H, m), 4.01(1H, m), 2.90-2.70 (2H, m), 1.48-1.32 (21H, m).

¹³C NMR (75 MHz, CDCl₃): δ=184.85, 169.91, 168.91, 155.84, 141.91,139.12, 131.41, 128.86, 128.13, 127.18, 124.99, 103.56, 103.13, 82.76,80.23, 72.15, 72.00, 57.64, 47.62, 42.71, 42.53, 32.02, 31.11, 28.44,28.02, 17.03, 14.33.

The protected (±)-Ketorolac L-threonine ester SPI003101 (13.50 g, 26.33mmol) was dissolved in trifluoroacetic acid (50 mL) under an argonatmosphere, at room temperature. The mixture was allowed to stir for 7hours at room temperature under an argon atmosphere. The brown solutionwas concentrated under reduced pressure and dried under high vacuum atroom temperature until a constant weight was achieved. The remainingbrown solid (10.2 g) was stirred in acetone (250 mL) at room temperaturefor 3 hours. The white precipitate that formed was filtered and driedunder high vacuum. The remaining solid (5.70 g) was dissolved in aminimal amount of DIUF water (5-10 mL) and a 1:1 mixture ofacetonitrile-acetone (100 mL) was added drop-wise over 1 hour whilestirring at room temperature. After the addition was complete, themixture was stored for 2 hours at room temperature. The whiteprecipitate that formed was filtered and dried under high vacuum at roomtemperature to a constant weight. The white solid (3.0 g) was purified afinal time by dissolving in DIUF water (5 mL). Most of the water wasremoved under reduced pressure to generate a thick, clear oil. Acetone(100 mL) was added to the oil in a drop-wise fashion over 30 minuteswhile stirring under an argon atmosphere. The mixture was stored for 3hours at −10° C. The precipitate was filtered and dried under highvacuum at room temperature until the weight was constant. The experimentproduced S(−)-Ketorolac-L-threonine ester, hydrochloride SPI0031A (2.32g, 22.4% yield based on SPI003101, 98.12% purity by HPLC) as whitesolid.

¹H NMR (300 MHz, DMSO): δ=8.80 (3H, br s), 7.73 (2H, d, J=7.5 Hz),7.61-7.46 (3H, m), 6.77 (1H, d, J=3.9 Hz), 6.16 (1H, d, J=3.9 Hz), 5.33(1H, m), 4.42-4.22 (4H, m), 2.76 (2H, m), 1.35 (3H, d, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=183.41, 169.64, 168.19, 142.32, 138.59,131.44, 128.35, 128.26, 126.22, 124.27, 103.18, 68.84, 55.31, 47.34,41.83, 30.18, 16.59.

CHN Analysis:

calc.: C, 58.09; H, 5.39; N, 7.13, Cl 9.02 (C₁₉H₂₁ClN₂O₅); found: C,58.61; H, 5.26; N, 7.10; Cl 8.16.

HPLC Analysis:

98.12% purity, r.t.=19.617 min, sample dissolved in DIUF water/ACN, 50%DIUF water (0.1% TFA)/50% ACN, Gemini C18 (#262049-2), 5u, 250×4.6 mm, 1mL/min., 37° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −108 deg (25° C., 52.5 mg/5 mL water, 589 nm)

Melting Point: 155-157° C. (decomposed)

Large negative specific rotation is consistent with the S(−)Ketorolacmoiety.

Synthesis of Fibric Acid Threonine Derivatives

Overview:

The procedure for the synthesis of the L-threonine esters of fenofibricacid is outlined in the Synthetic Sequence section. The completeprocedure and analytical data is given in the Experimental Section. Ingeneral, fenofibric acid (100 g batches) was prepared from4-chloro-4′-hydroxybezophenone in accordance with the known procedure.Fenofibric acid was coupled with the t-butyl esters of N-boc protectedamino acid (L-threonine) using EDC as the coupling agents and acatalytic amount of DMAP. The protecting groups were removed at lowtemperature (5° C., 3-6 days) with a mixture of hydrochloric acid inacetic acid (1M) with dichloromethane. The amino acid ester salts offenofibric acid were purified by crystallization from ethyl acetate,dried under high vacuum, and shipped to Signature Pharmaceuticals Inc.,after analysis by NMR, HPLC, CHN, and melting point. This procedure wasrepeated substituting L-serine and L-hydroxyproline for L-Threonine forcomparative purposes.

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters offenofibric acid: a) Boc-Ser-OtBu, EDC, DMAP, CH₂Cl₂; b) Boc-Thr-OtBu,EDC, DMAP, CH₂Cl₂; c) Boc-Hyp-OtBu, EDC, DMAP, CH₂Cl₂; d) HCl, AcOH,CH₂Cl₂.Experimental Section:

The synthesis of SPIB00201, SPIB00202 and SPIB00203 was conducted in oneor two batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

Synthesis of Fenofibric Acid:

A mixture of 4-chloro-4′-hydroxybezophenone (116 g, 0.500 mole) andsodium hydroxide (120 g, 3.00 mole) in acetone (1 L) was heated toreflux for 2 hours. The heating was stopped and the heating source wasremoved. A mixture of chloroform (179 g, 1.50 mole) in acetone (300 mL)was added drop-wise. The reaction mixture was stirred overnight withoutheating. The mixture was heated to reflux for 8 hours and then allowedto cool to room temperature. The precipitate was removed by filtrationand washed with acetone (100 mL). The filtrate was concentrated underreduced pressure to give a brown oil. Water (200 mL) was added to thebrown oil and was acidified (to pH=1) with 1N hydrochloric acid. Theprecipitate, which formed was filtered and dried under high vacuum. Theremaining yellow solid (268 g) was recrystallized from toluene in 4batches (400 mL toluene each). After filtration and drying under highvacuum, the experiment produced fenofibric acid (116 g, 73% yield) as alight yellow solid.

¹H NMR (300 MHz, DMSO-d₆): δ=13.22 (1H, s, br), 7.72 (4H, d, J=8.4 Hz),7.61 (2H, d, J=7.8 Hz), 6.93 (2H, d, J=7.8 Hz), 1.60 (6H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=192.96, 174.18, 159.35, 136.84, 136.12,131.67, 131.02, 129.12, 128.43, 116.91, 78.87, 25.13.

2) SPIB00201: L-serine-fenofibric acid ester

To a mixture of fenofibric acid (11.6 g, 36.3 mmol),N-carbobenzyloxy-L-serine t-butyl ester (Boc-Ser-OtBu, 8.62 g, 33.0mmol), EDC (7.59 g, 39.6 mmol), and DMAP (484 mg, 3.96 mmol) cooled inan ice-water bath was added anhydrous dichloromethane (150 mL) dropwise.After addition was complete, the ice bath was removed and the reactionmixture was stirred under an argon atmosphere at room temperature for 20hours. After 20 hours, the additional dichloromethane (200 mL) was addedand the solution was washed with water (2×200 mL) and brine (200 mL).After drying over sodium sulfate and filtration, the solution wasconcentrated under reduced pressure. The remaining yellow oil (21.2 g)was purified by column chromatography on silica gel (400 g, 0.035-0.070mm, 6 nm pore diameter), eluting with heptane/ethyl acetate (3:1). Afterconcentration of the product-containing fractions under reduced pressureand drying under high vacuum until the weight was constant, theexperiment produced the protected L-serine-fenofibric acid esterSPIB0020101 (16.2 g, 87% yield) as a light yellow oil.

¹H NMR (300 MHz, CDCl₃): δ=7.75 (2H, d, J=9.0 Hz), 7.72 (2H, d, J=9.0Hz), 7.45 (2H, d, J=8.7 Hz), 6.86 (2H, d, J=8.7 Hz), 5.04 (1H, d, J=6.9Hz), 4.55-4.42 (3H, m), 1.66 (3H, s), 1.65 (3H, s), 1.43 (9H, s), 1.39(9H, s).

¹³C NMR (75 MHz, CDCl₃): δ=193.92, 172.99, 168.07, 159.24, 154.87,138.24, 136.19, 131.94, 131.06, 130.40, 128.41, 117.26, 82.88, 80.13,79.24, 65.44, 53.44, 28.27, 27.92, 25.70, 25.30.

To a stirred solution of the protected L-serine-fenofibric acid esterSPIB0020101 (16.2 g, 28.8 mmol) in anhydrous dichloromethane (100 mL)cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (400 mL, 1M, 400 mmol) drop-wise. Thereaction mixture stirred for 3 days at 5° C. After three days themixture was concentrated under reduced pressure and dried under highvacuum to remove acetic acid. To the remaining light yellow oil (24.7 g)was added ethyl acetate (100 mL). The solution was concentrated anddried a second time. To the remaining light yellow oil (17.0 g) wasadded ethyl acetate (65 mL). The mixture was heated to reflux for 5minutes and cooled to room temperature. The precipitate was removed byfiltration and dried under high vacuum overnight at room temperature,then at 43° C. for one hour. The experiment produced theL-serine-fenofibric acid ester, hydrochloride SPIB00201 (7.66 g, 60%yield) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.12 (1H, s, br), 8.77 (3H, s, br), 7.72(4H, m), 7.62 (2H, d, J=8.4 Hz), 6.92 (2H, d, J=9.0 Hz), 4.62 (1H, dd,J=12.0, 4.2 Hz), 4.50 (1H, dd, J=12.0, 2.4 Hz), 4.41 (1H, m), 1.64 (3H,s), 1.63 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.06, 171.70, 168.06, 158.72, 136.93,136.06, 131.73, 131.09, 129.62, 128.49, 117.64, 79.02, 62.99, 51.11,25.04, 24.94.

HPLC Analysis:

100% purity; r.t.=4.361 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3 C,Luna C18, serial # 167917-13; 20 ul inj., NB275-49.

CHN Analysis:

calc.: C, 54.31; H, 4.79; N, 3.17; found: C, 54.37; H, 4.78; N, 3.12.

Melting point: 151° C. (dec.)

SPIB00202: L-threonine-fenofibric acid ester

To a mixture of fenofibric acid (25.5 g, 79.9 mmol),N-carbobenzyloxy-L-threonine t-butyl ester (Boc-Thr-OtBu, 20.0 g, 72.6mmol), EDC (16.7 g, 87.1 mmol), and DMAP (1.06 g, 8.71 mmol) cooled inan ice-water bath was added anhydrous dichloromethane (200 mL),dropwise. After the addition was complete, the ice bath was removed andthe reaction mixture was stirred under an argon atmosphere at roomtemperature for 20 hours. After 20 hours, additional EDC (1.39 g, 7.26mmol) was added and the experiment was allowed to stir over the weekendat room temperature under an argon atmosphere. After 4 days, additionaldichloromethane (300 mL) was added and the solution was washed withwater (300 mL) and brine (300 mL). After drying over sodium sulfate andfiltration, the solution was concentrated under reduced pressure. Theremaining yellow oil (53.5 g) was purified by column chromatography onsilica gel (500 g, 0.035-0.070 mm, 6 nm pore diameter), eluting withheptane/ethyl acetate (3:1). After concentration of theproduct-containing fractions under reduced pressure and drying underhigh vacuum until the weight was constant, the experiment produced theprotected L-threonine-fenofibric acid ester SPIB0020201 (34.1 g, 82%yield) as a white foam.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (2H, d, J=8.4 Hz), 7.72 (2H, d, J=8.4Hz), 7.45 (2H, d, J=8.4 Hz), 6.87 (2H, d, J=8.4 Hz), 5.47 (1H, m), 4.98(1H, d, J=9.9 Hz), 4.31 (1H, d, J=9.9 Hz), 1.65 (3H, s), 1.64 (3H, s),1.45 (9H, s), 1.42 (9H, s), 1.22 (3H, d, J=6.3 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=193.94, 172.14, 168.70, 159.26, 155.62,138.28, 136.18, 131.90, 131.08, 130.37, 128.43, 117.40, 82.70, 80.17,79.38, 72.02, 57.46, 28.30, 27.99, 26.44, 24.79, 16.90.

To a stirred solution of the protected L-threonine-fenofibric acid esterSPIB0020201 (34.1 g, 59.2 mmol) in anhydrous dichloromethane (100 mL)cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (600 mL, 1M, 600 mmol) drop-wise. Thereaction mixture was kept for 6 days at 5° C. The mixture wasconcentrated under reduced pressure and dried under high vacuum toremove acetic acid. To the remaining white solid (45.8 g) was addedethyl acetate (500 mL). The mixture was heated to reflux for 10 minutesand cooled to room temperature. The precipitate was removed byfiltration and dried under high vacuum overnight at room temperature.The experiment produced the L-threonine-fenofibric acid ester,hydrochloride SPIB00202 (26.3 g, 97% yield) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.10 (1H, s, br), 8.84 (3H, s, br), 7.73(4H, m), 7.63 (2H, d, J=8.1 Hz), 6.89 (2H, d, J=8.7 Hz), 5.44 (1H, m),4.31 (1H, s), 1.64 (3H, s), 1.62 (3H, s), 1.38 (3H, d, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.04, 171.00, 168.13, 158.76, 136.90,136.08, 131.70, 131.06, 129.49, 128.48, 117.41, 78.99, 69.40, 55.21,25.59, 24.22, 16.06.

HPLC Analysis:

98.59% purity; r.t.=4.687 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3C, Luna C18, serial # 167917-13; 20 ul inj., NB275-49, DAD1 B,Sig=210.4, Ref=550,100.

CHN Analysis:

calc.: C, 55.27; H, 5.08; N, 3.07; found: C, 54.98; H, 5.13; N, 3.03.

Melting point: 160.5° C. (dec.)

SPIB00203: L-hydroxyproline-fenofibric acid ester

To a mixture of fenofibric acid (24.9 g, 78.1 mmol),N-carbobenzyloxy-L-hydroxyproline t-butyl ester (Boc-Hyp-OtBu, 20.4 g,71.0 mmole), EDC (16.3 g, 85.2 mmol), and DMAP (1.04 g, 8.52 mmol)cooled in an ice-water bath was added anhydrous dichloromethane (200 mL)dropwise. After the addition was complete, the ice bath was removed andthe reaction mixture was stirred under an argon atmosphere at roomtemperature for 20 hours. After 20 hours, additional EDC (1.63 g, 8.52mmol) was added and the experiment was allowed to stir over the weekendat room temperature under an argon atmosphere. After 4 days the solutionwas washed with water (200 mL) and brine (200 mL). After drying oversodium sulfate and filtration, the solution was concentrated underreduced pressure. The remaining yellow oil (49.4 g) was purified bycolumn chromatography on silica gel (500 g, 0.035-0.070 mm, 6 nm porediameter), eluting with heptane/ethyl acetate (2:1). After concentrationof the product containing fractions under reduced pressure and dryingunder high vacuum until the weight was constant, the experiment producedthe protected L-hydroxyproline-fenofibric acid ester SPIB0020301 (26.4g, 63% yield) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.76 (2H, d, J=8.1 Hz), 7.73 (2H, d, J=8.1Hz), 7.46 (2H, d, J=8.1 Hz), 6.84 (2H, d, J=8.1 Hz), 5.32 (1H, m), 4.13(0.38H, t, J=7.8 Hz), 4.00 (0.62H, t, J=7.8 Hz), 3.67 (1.62H, m), 3.46(0.38H, d, J=12.6 Hz), 2.29 (1H, m), 2.15 (1H, m), 1.68 (3H, s), 1.66(3H, s), 1.44-1.38 (18H, m).

¹³C NMR (75 MHz, CDCl₃): δ=193.88, 172.98, 171.14, 159.25, 153.48,138.23, 136.16, 131.99, 131.08, 130.36, 128.44, 117.03, 116.91, 81.48,80.32, 80.20, 79.19, 74.03, 73.26, 58.23, 51.88, 51.58, 36.33, 35.31,31.92, 28.29, 28.00, 25.89, 24.95.

To a stirred solution of the protected L-hydroxyproline-fenofibric acidester SPIB0020301 (26.0 g, 44.2 mmol) in anhydrous dichloromethane (100mL) cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (450 mL, 1M, 450 mmol) drop-wise. Thereaction mixture stirred for 4 days at 5° C. After four days the mixturewas concentrated under reduced pressure and dried under high vacuum toremove acetic acid. To the remaining yellow oil (31.5 g) was added ethylacetate (200 mL). The mixture was sonicated and then concentrated underreduced pressure and dried under high vacuum. To the remaining whitesolid (23.2 g) was added ethyl acetate (300 mL). The ethyl acetatemixture was heated to reflux for 10 minutes and cooled to roomtemperature. The precipitate was removed by filtration and dried underhigh vacuum overnight at room temperature. The experiment produced theL-hydroxyproline-fenofibric acid ester, hydrochloride SPIB00203 (15.8 g,76% yield) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.07 (1H, s, br), 10.75 (1H, s, br), 9.40(1H, s, br), 7.71 (4H, d, J=8.1 Hz), 7.60 (2H, d, J=8.1 Hz), 6.96 (2H,d, J=8.1 Hz), 5.42 (1H, m), 4.24 (1H, t, J=9.0 Hz), 3.61 (1H, dd,J=13.2, 4.2 Hz), 3.28 (1H, d, J=13.2 Hz), 2.35 (2H, m), 1.66 (3H, s),1.64 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.00, 171.52, 169.14, 158.81, 136.87,136.09, 131.81, 131.05, 129.48, 128.46, 117.28, 78.99, 73.79, 57.54,50.23, 34.13, 25.69, 24.49.

HPLC Analysis:

100% purity; r.t.=8.369 min.; 60% DIUF water (0.1% TFA)/40%acetonitrile; 1 mL/min; 36.4 C; Luna C18, 5u column (serial # 191070-3),4.6×250 mm; 20 ul injection; DAD1 A, Sig=210.4, Ref=550,100.

HPLC-MS (ESI): calculated: M⁺=431; found M+H=432.3

Melting point: 187.5° C. (dec.)

Overview: L-Threonine Derivative of Acetylsalkyelic Acid

The procedure for the synthesis of the L-Threonine of acetylsalicylicacid is outlined in the Synthetic Sequence section. The completeprocedure and analytical data is given in the Experimental Section. Ingeneral, acetylsalicyloyl chloride (10 g-25 g, in batches) was coupledwith the N-benzyloxy/benzyl ester protected amino acids in the presenceof pyridine. Once the reactions were complete (24 to 48 hours at roomtemperature), the mixture was poured into ice-cold 2N hydrochloric acid.The dichloromethane fraction was then washed with sodium bicarbonate,water and brine. After drying over sodium sulfate, filtration, andconcentration the crude protected amino acid esters of acetylsalicylicacid were purified by flash chromatography on silica gel. The proceduregenerated the protected amino acid esters of acetylsalicylic acid inyields ranging from 68% to 95%. The protecting groups were removed byhydrogenation (20 psi H₂) in the presence of 10% palladium on carbon.The amino acid esters of acetylsalicylic acid were extracted away fromthe palladium catalyst with water, concentrated, and dried. The finalcompounds were washed with solvent (water, dioxane, acetonitrile, and/ordichloromethane) until pure and dried under high vacuum until a constantweight was achieved. The L-serine and the L-hydroxyproline esters wereprepared for comparative purposes.

Synthesis of the L-serine, L-threonine, and L-Hydroxyproline esters ofacetylsalicylic acid: a) pyridine, CH₂Cl₂; b) 10% Pd/C, EtOH, EtOAc.

Experimental Section:

The synthesis of SPIB00101, SPIB00102 and SPIB00103 was conducted in oneor two batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinckrodt.

SPIB00102: 2-O-Acetylsalicylic acid (2S,3R)-(−)-threonine ester

A mixture of N-carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl,21.77 g, 63.40 mmole) and pyridine (25 mL) in anhydrous dichloromethane(500 mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (17.63 g, 88.76 mmole) was added and themixture was allowed to warm to room temperature and stir overnight.After 24 hours, the mixture was poured into ice-cold 2N hydrochloricacid (400 mL). After mixing, the layers were separated and thedichloromethane fraction was washed with water (500 mL), saturatedsodium bicarbonate solution (500 mL), water (500 mL), brine (500 mL) anddried over sodium sulfate (25 g). After filtration, concentration underreduced pressure, and drying under high vacuum, the remaining yellow oil(35.43 g) was purified by flash chromatography on silica gel (300 g,0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate(3:1). After concentration of the product containing fractions underreduced pressure and drying under high vacuum until the weight wasconstant, the experiment produced the protectedacetylsalicylic-L-threonine ester SPIB0010201 (28.1 g, 88% yield) as acolorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (1H, d, J=7.5 Hz), 7.51 (1H, dt, J=7.5,1.5 Hz), 7.34-7.17 (11H, m), 7.06 (1H, d, J=7.2 Hz), 5.62 (2H, m), 5.13(4H, m), 4.65 (1H, dd, J=9.6, 2.4 Hz), 2.29 (3H, s), 1.38 (3H, d, J=6.6Hz).

¹³C NMR (75 MHz, CDCl₃): δ=169.35, 169.22, 162.73, 156.26, 150.41,135.79, 134.67, 133.77, 131.24, 128.35, 128.24, 128.08, 127.95, 125.78,123.51, 122.61, 71.22, 67.72, 67.26, 57.64, 20.98, and 16.88.

The protected acetylsalicylic-L-threonine ester SPIB0010201 (14.50 g,28.68 mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100mL) at room temperature and added to a Parr bottle that contained 10%palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (20 psi). After 20hours of shaking, the palladium catalyst was removed by filtrationthrough celite. The remaining solids (palladium/celite and product) werewashed with water (600×4 mL) until the product was removed. The ethanoland water fractions were concentrated under reduced pressure at roomtemperature. The remaining solids were washed with water (20 mL) anddioxane (20 mL) for 48 hours. After filtration, the remaining whitesolid was dried at room temperature under high vacuum until the productweight was constant (16 hours). The experiment producedacetylsalicylic-L-threonine ester, SPIB00102 (4.40 g, 55% yield) as awhite solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.00 (1H, dd, J=7.8, 1.5 Hz), 7.74 (1H, dt,J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8, 1.5 Hz), 7.27 (1H, dd, J=7.8, 1.5Hz), 5.76 (1H, dq, J=6.9, 3.0 Hz), 4.49 (1H, d, J=3.0 Hz), 2.39 (3H, s),1.55 (3H, d, J=6.9 Hz).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.03, 168.84, 163.97, 149.56, 135.32,131.26, 126.85, 123.48, 121.49, 69.16, 56.36, 20.45, and 15.86.

HPLC Analysis:

98.7% purity; r.t=6.233 min; Luna C18 5u column (sn 167917-13); 4.6×250mm; 254 nm; 35% MeOH/65% TFA (0.1%) pH=1.95; 35 C; 20 ul inj.; 1 ml/min;sample dissolved in mobile phase with 1 drop phosphoric acid.

CHN Analysis:

calc.: C 55.51, H 5.38, and N 4.98; found: C 55.37, H 5.40, and N 5.03.

Melting point: 153.5° C. (dec.)

SPIB00101: 2-O-Acetylsalicylic acid (2S)-(+)-serine ester

A mixture of N-carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl, 23.17g, 70.34 mmole) and pyridine (30 mL) in anhydrous dichloromethane (500mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (21.07 g, 106.1 mmole) was added and themixture was allowed to warm to room temperature and stir over two days.After 48 hours, the mixture was poured into ice-cold 2N hydrochloricacid (400 mL). After mixing, the layers were separated and thedichloromethane fraction was washed water (500 mL), saturated sodiumbicarbonate solution (500 mL), water (500 mL), brine (500 mL) and driedover sodium sulfate (25 g). After filtration, concentration underreduced pressure, and drying under high vacuum, the remaining brownsolid (47.19 g) was purified by flash chromatography on silica gel (200g, 0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethylacetate (3:1). After concentration of the product containing fractionsunder reduced pressure and drying under high vacuum until the weight wasconstant, the protected acetylsalicylic-L-serine ester SPIB0010101(32.97 g, 95% yield) was produced as a white solid.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (1H, d, J=7.8 Hz), 7.55 (1H, dt, J=7.8,1.5 Hz), 7.33-7.21 (11H, m), 7.08 (1H, d, J=7.5 Hz), 5.68 (1H, d, J=8.4Hz), 5.20 (2H, s), 5.12 (2H, s), 4.77 (1H, m), 4.66 (1H, dd, J=11.4, 3.3Hz), 4.57 (1H, dd, J=11.4, 3.3 Hz), 2.30 (3H, s).

¹³C NMR (75 MHz, CDCl₃): δ=169.45, 169.09, 163.68, 163.35, 155.57,150.77, 135.87, 134.75, 134.07, 131.44, 128.50, 128.43, 128.27, 128.14,128.04, 125.92, 123.71, 122.18, 67.83, 67.27, 64.63, 53.55, and 21.03.

The protected acetylsalicylic-L-serine ester SPIB0010101 (21.0 g, 42.7mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100 mL) atroom temperature and added to a Parr bottle that contained 10% palladiumon carbon (4.20 g, 50% wet) under a nitrogen atmosphere. The nitrogenatmosphere was replaced with hydrogen gas (20 psi). After 5 hoursadditional 10% palladium catalyst (4.26 g) was added and the hydrogenatmosphere was returned (20 psi). After an additional 20 hours ofshaking at room temperature, the palladium catalyst was removed byfiltration through celite. The remaining solids (palladium/celite andproduct) were washed with water (1500×2 mL) until the product wasremoved. The ethanol and water fractions were concentrated under reducedpressure at room temperature. The remaining solid (7.17 g) was dissolvedin DIUF water (4.3 L), filtered through celite to remove insolublematerial, and concentrated under high vacuum at room temperature. Thewhite solid was then washed with 1,4-dioxane (100 mL) and DIUF water (50mL) overnight. After 24 hours the solid was filtered and dried underhigh vacuum until the weight was constant (24 hours).

The experiment produced the acetylsalicylic-L-serine ester SPIB00101(6.17 g, 54% yield) as a white solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.05 (1H, dd, J=7.8, 1.5 Hz), 7.75 (1H, dt,J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8, 0.9 Hz), 7.27 (1H, dd, J=7.8, 0.9Hz), 4.87 (1H, dd, J=12.6, 4.2 Hz), 4.79 (1H, dd, J=12.6, 3.0 Hz), 4.62(1H, dd, J=4.2, 3.0 Hz), 2.39 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.01, 168.58, 164.54, 149.72, 135.39,131.59, 126.87, 123.62, 121.15, 62.38, 52.05, and 20.44.

HPLC Analysis:

98.1% purity; r.t.=5.839 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35C; Luna C18, 3u column (SN 184225-37), 4.6×250 mm; 22 ul injection;DAD1B, Sig=240, 4 Ref=550,100.

CHN Analysis:

calc.: C, 53.93; H, 4.90; and N, 5.24. found: C, 54.02; H, 5.00; and N,5.23.

Melting point: 147.0° C. (dec.)

SPIB00103: 2-O-Acetylsalicylic acid (2S,4R)-4-hydroxyproline ester

A mixture of N-carbobenzyloxy-L-Hydroxyproline benzyl ester (Z-Ser-OBzl,21.5 g, 60.5 mmole) and pyridine (25 mL) in anhydrous dichloromethane(500 mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (13.2 g, 66.6 mmole) was added and the mixturewas allowed to warm to room temperature and stir overnight. After 24hours, additional acetylsalicyloyl chloride (5.0 g, 25.2 mmole) wasadded and the mixture was allowed to stir overnight. After 48 hours, themixture was poured into ice-cold 1N hydrochloric acid (500 mL). Aftermixing, the layers were separated and the dichloromethane fraction waswashed with water (500 mL), saturated sodium bicarbonate solution (500mL), water (500 mL), brine (500 mL) and dried over sodium sulfate (25g). After filtration, concentration under reduced pressure, and dryingunder high vacuum, the remaining yellow oil (40.7 g) was purified byflash chromatography on silica gel (460 g, 0.035-0.070 mm, 6 nm porediameter), eluting with heptane/ethyl acetate (3:1). After concentrationof the product containing fractions under reduced pressure and dryingunder high vacuum until the weight was constant, the the protectedacetylsalicylic-L-Hydroxyproline ester SPIB0010301 (21.31 g, 68% yield)was produced as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.92 (1H, d, J=7.8 Hz), 7.56 (1H, t, J=7.8Hz), 7.34-7.21 (10H, m), 7.09 (1H, d, J=7.8 Hz), 5.48 (1H, s), 5.21 (2H,m), 5.03 (2H, d, J=15 Hz), 4.57 (1H, m), 3.85 (2H, m), 2.53 (1H, m),2.28 (4H, m).

¹³C NMR (75 MHz, CDCl₃): δ=171.72, 171.49, 169.25, 163.47, 163.30,154.52, 153.93, 150.54, 136.05, 135.94, 135.21, 135.00, 134.17, 134.12,128.43, 128.32, 128.28, 128.20, 128.05, 127.98, 127.94, 127.79, 125.89,123.70, 122.46, 122.38, 73.24, 72.59, 67.33, 67.11, 66.97, 58.02, 57.69,52.47, 52.15, 36.74, 35.65, 20.90.

The protected acetylsalicylic-L-Hydroxyproline ester SPIB0010301 (10.6g, 20.5 mmole) was dissolved in ethanol (75 mL) and ethyl acetate (75mL) at room temperature and added to a Parr bottle that contained 10%palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (20 psi). After 17hours of shaking at room temperature, the reaction mixture was washedwith water (500 mL) for two hours. The organic layer (top) was removedvia pipette and the aqueous layer was filtered through celite. The waterfraction was concentrated under reduced pressure at room temperature.The remaining solid (6.71 g) was then washed with anhydrousdichloromethane (35 mL) overnight. After 24 hours the solid was filteredand dried under high vacuum until the weight was constant (24 hours).The acetylsalicylic-L-Hydroxyproline ester, SPIB00301 (2.87 g, 47.7%yield) was produced as a white solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.09 (1H, d, J=7.5 Hz), 7.75 (1H, t, J=7.5Hz), 7.48 (1H, t, J=7.5 Hz), 7.28 (1H, d, J=7.5 Hz), 5.69 (1H, m), 4.76(1H, t, J=7.5 Hz), 3.86 (1H, dd, J=13.5, 3.9 Hz), 3.74 (1H, d, J=13.5Hz), 2.81 (1H, dd, J=15.0, 7.5 Hz), 2.60 (1H, m), 2.40 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.13, 170.25, 164.31, 149.65, 135.36,131.54, 126.87, 123.54, 121.37, 73.86, 58.34, 50.95, 34.38, and 20.48.

HPLC Analysis:

98.3% purity; r.t.=7.201 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35C; Luna C18, 3u column (SN 184225-37), 4.6×250 mm; 22 ul injection;DAD1B, Sig=240, 4 Ref=550,100.

CHN Analysis:

calc.: C, 57.34; H, 5.16; and N, 4.78. found: C, 57.09; H, 5.23; and N,4.91.

Melting point: 162° C. (dec.)

Specificity of L-Threonine to Separate Racemic and StereoisomericMixtures:

When the racemic Ibuprofen, racemic Ketoprofen, racemic Ketorolac werereacted with L-Threonine, the resulting racemic drug-L-Threonine estershad quite varying physicochemical properties, and it was easy tocrystallize or precipitate from the reaction mixture. One trained in theart of making amino acid esters of such drug would conclude that Hydroxyproline and Serine esters of these drugs will also result in separationof the reacemic mixtures in the form of corresponding esters. Theinventor surprisingly found that only L-Threonine ester are readilyseparable and it is not the same with other amino acids. In case of bothHydroxyproline and Serine esters of Ketoprofen, the resulting KetoprofenHydroxyproline ester or serine ester were not as readily separable likeL-Threonine ester of Ketoprofen. Only L-Threonine ester was able toseparate the racemic mixture of Ketoprofen into the individualenantiomeric esters. The same surprising results were obtained forKetorolac as well.

Efficacy (Anti Nociceptive Potential) of Synthesis of the L-serine,L-Threonine, and L-hydroxyproline esters of (±)-Ibuprofen by EmployingAcetylcholine Induced Abdominal Constriction Method in Male Albino Mice:

The present study was conducted to evaluate the efficacy of L-serine,L-Threonine, and L-hydroxyproline esters of (±)-Ibuprofen taking intoaccount the antagonizing property on acetylcholine induced writhe as anindex in albino mice. Ibuprofen (racemic mixture) and ibuprofen (S)-(+)served as reference controls.

Different new formulations of ibuprofen and reference controls viz.,ibuprofen (racemic mixture) and ibuprofen (S)-(+) were administered bygavage to male albino mice (Swiss strain), using 5% (v/v) Tween 80 inmilli Q water as the vehicle. The study was conducted at two dose levelsviz. 50 mg and 100 mg/kg body weight along with a vehicle control group.At each dose level 10 animals were used. All the doses were expressed asibuprofen molar equivalents. The doses used as well as the molarequivalents are presented below. TABLE 2 Formulation: Molar Equivalent:Formulation Molar equivalent S-(+)-Ibuprofen-L-Threonine ester 0.833units are equivalent to 1 unit of Ibuprofen (±)-Ibuprofen-L-serine ester1.6 units are equivalent to 1 unit of Ibuprofen(±)-Ibuprofen-L-hydroxyproline 1.55 units are equivalent to 1 unit ofester Ibuprofen

TABLE 3 Test Item: Group: Dose(mg/kg): Equivalent wt. Of the test item:Equivalent Dose (mg per kg) weight of the [in terms of Test item TestItem Group Ibuprofen] [mg/kg] Vehicle Vehicle control 0.0 — GroupS-(+)-Ibuprofen-L- Test Group 1 50.0 41.65 Threonine ester Test Group 2100.0 83.30 (±)-Ibuprofen-L- Test Group 3 50.0 80.0 serine ester TestGroup 4 100.0 160.0 (Ibuprofen S) (±)-Ibuprofen-L- Test Group 5 50.077.5 hydroxyproline ester Test Group 6 100.0 155.0 Ibuprofen (racemicTest Group 7 50.0 50.0 mixture) Test Group 8 100.0 100.0 Ibuprofen S+Test Group 9 50.0 25.0 Test Group 10 100.0 50.0

The efficacy in terms of antagonizing effect on acetylcholine inducedsingle writhe at two dose levels—50.0 and 100.0 mg/kg for the threeformulations and reference controls are presented below. TABLE 4 TestItem: Group: Dose (mg/kg): Number of animals showing absence of singlewrithe (out of 10) Number of animals showing absence of single writhe(number of animals Dose (mg per dose = 10) per kg) One hour [in terms ofafter Three hours Test Item Group Ibuprofen] dosing after dosing VehicleVehicle 0.0 0 0 control S-(+)- Low dose 50.0 1 0 Ibuprofen-L High dose100.0 3 0 Threonine ester (±)- Low dose 50.0 4 2 Ibuprofen-L- High dose100.0 6 4 serine ester (±)- Low dose 50.0 5 4 Ibuprofen-L- High dose100.0 7 7 hydroxyproline ester Ibuprofen Low dose 50.0 4 2 (racemic Highdose 100.0 6 6 mixture) Ibuprofen S+ Low dose 50.0 5 1 High dose 100.0 66

Statistical analysis employing Chi-square test procedure did not showany statistically significant difference among the formulations incomparison to reference control, while comparing the number of animalsnot showing writhe in each groups, as the respective “p” was found to begreater than 0.05, the level of significance. Also note that the dose ofS(+)Ibuprofen-L-Threonine ester is only ½ the normal dose of Ibuprofen.

From clinical observation based on the number of animals not showingwrithes due to administration of acetylcholine,(±)-Ibuprofen-L-hydroxyproline ester was found to be more effective inantagonizing the acetylcholine induced writhe when compared to otherformulations and Ibuprofen (racemic) and Ibuprofen (S)-(+). TABLE 5Summary of Efficacy of L-serine, L-Threonine, and L- hydroxyprolineesters of (±)-Ibuprofen, Ibuprofen (racemic mixture) and Ibuprofen(S)-(+)-Based on Antagonizing Property of Acetylcholine Induced Writhein Albino Mice Number of animals showing Dose absence of single writhe(number (mg/kg) of animals per dose = 10) [in terms of One hour afterThree hours Ibuprofen] Test Item dosing after dosing 50 mg/kg Vehiclecontrol 0 0 S-(+)-Ibuprofen-L- 1 0 Threonine ester (±)-Ibuprofen-L- 4 2serine ester (±)-Ibuprofen-L- 5 4 hydroxyproline ester Ibuprofen 4 2(racemic mixture) Ibuprofen (S)-(+) 5 1

TABLE 6 100 mg/kg Vehicle control 0 0 S-(+)-Ibuprofen- 3 0 L-Threonineester (±)-Ibuprofen-L- 6 4 serine ester (±)-Ibuprofen-L- 7 7hydroxyproline ester Ibuprofen 6 6 (racemic mixture) Ibuprofen(S)-(+) 66

The data were subjected to statistical analysis employing Chi-squaretest procedure for evaluating the efficacy of the new formulations incomparison to the reference controls. The test did not show anystatistically significant difference among the formulations incomparison to reference control, while comparing the number of animalsnot showing writhe in each groups, as the respective “p” was found to begreater than 0.05, the level of significance.

The data is also summarized in FIGS. 1 and 2. From clinical observationsand bar diagram for comparative efficacy (FIGS. 1 and 2) based on thenumber of animals not showing writhes due to administration ofacetylcholine, (±)-Ibuprofen-L-hydroxyproline ester was found to be moreeffective in antagonizing the acetylcholine induced writhe when comparedto other formulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).However, as shown hereinbelow, the hydroxyproline ester was found to bemore toxic than the threonine ester, as shown in the 28 day chronictoxicity study described hereinbelow. Thus, the therapeutic index forthe L-Threonine ester is signifignatly more favorable than with theother esters of hydroxy containing amino acids.

CONCLUSION

The present study was conducted to evaluate the relative efficacy of newformulations of ibuprofen. For this the antagonizing property of newformulations on acetylcholine writhes was taken as an index to determinethe relative efficacy of the formulations. Ibuprofen (racemic mixtureand ibuprofen (S)-(+) served as reference controls. The study wasconducted at two dose levels (50.0 and 100.0 mg/kg) along with a vehiclecontrol group.

Gastric Mucosal Irritation Potential of L-serine, L-Threonine, andL-hydroxyproline esters of (±)-Ibuprofen in Fasted Male Albino Rats

Summary

The present study was conducted to determine the relative potential ofnew formulations of ibuprofen (L-serine, L-Threonine, andL-hydroxyproline esters of (±)-Ibuprofen) to cause gastric mucosalirritation/lesions in fasted male albino rats. Ibuprofen (racemicmixture) and lbuprofen(S)-(+) served as reference controls.

Different new formulations of ibuprofen and ibuprofen (racemic mixture)and ibuprofen(S)-(+) were administered by gavage to fasted male albinorats (Wistar strain), using 5% solution of Tween 80 in milli Q water asthe vehicle. The study was conducted at two dose levels viz. 200 mg and300 mg/kg body weight along with a vehicle control group. At each doselevel 5 animals were used. All the doses were expressed as ibuprofen(racemic mixture) molar equivalents. The doses used as well as the molarequivalents were presented below. TABLE 7 Formulation: Molar EquivalentFormulation Molar equivalent S-(+)-Ibuprofen-L-Threonine ester 0.833units are equivalent to 1 unit of Ibuprofen (±)-Ibuprofen-L-serine ester1.60 units are equivalent to 1 unit of Ibuprofen(±)-Ibuprofen-L-hydroxyproline 1.55 units are equivalent to 1 unit ofester Ibuprofen

The various groups used are tabulated hereinbelow: TABLE 8 Test item:group: Dose (mg/kg) Equivalent wt. Equivalent weight of Dose (mg per kg)the [in terms of Test item Test item Group Ibuprofen] [mg/kg] VehicleVehicle control 0.0 — Group S-(+)-Ibuprofen-L- Test Group 1 200.0 0.0Threonine ester Test Group 2 300.0 166.6 (±)-Ibuprofen-L-serine TestGroup 1 200.0 249.9 ester Test Group 2 300.0 320.0 (±)-Ibuprofen-L- TestGroup 1 200.0 480.0 hydroxyproline ester Test Group 2 300.0 310.0Ibuprofen (racemic Test Group 1 200.0 465.0 mixture) Test Group 2 300.0300.0 Ibuprofen (S)-(+) Test Group 1 200.0 100.0 Test Group 2 300.0150.0

The rats were fasted for a period of 18 to 22 hours before dosing. Thetest item was administered as a single dose by gavage. Three hours afterdrug administration, the animals were killed humanely by CO₂ gasinhalation. The stomach was dissected out and observed for

-   -   the quantity of mucous exudate,    -   degree of hyperemia and thickening of stomach wall,    -   hemorrhagic spots (focal or diffuse), nature of hemorrhages        (petechial or ecchymotic) along with the size and    -   perforations or any other lesions

The observations on gastric mucosal irritation of animals of variousgroups were summarized as follows: None of the animals in the Vehiclecontrol group, S(+)Ibuprofen-L-Threonine Ester (200 and 300 mg/kg),(±)Ibuprofen-L-Serine (200 and 300 mg/kg), (±)Ibuprofen-L-Hydroxyproline(200 and 300 mg/k) groups showed any evidence of gastric mucosalirritation. In the (±)Ibuprofen 200 mg/kg dose, 1 out of 5 animalsshowed evidence of gastric mucosal irration. In the case of (±)Ibuprofen300 mg/kg dose, 3 out of 5 animals showed severe gastric mucosalirritation. Surprisingly, in the S(+)Ibuprofen group at 200 mg/kg dose,all five animals dosed showed evidence of gastric mucosal irritation,and 3 out of 5 animals showed severe gastric mucosal irritation in theS(+)Ibuprofen group. Please note that the last group is pure enantiomer,S(+) variety.

The results of the present study showed that none of the formulations ofibuprofen had caused any evidence of irritation of gastric mucosa infasted male albino rats of male sex at the two dose levels tested (200mg and 300 mg/kg body weight). In contrast both ibuprofen (racemicmixture) and ibuprofen S(+) had caused irritation of gastric mucosa atthe two dose levels tested. Further ibuprofen S(+) was found to be moregastric mucosal irritant than ibuprofen racemic mixture.

28-Day Chronic Toxicity Studies with S(+) Ibuprofen L-Threonine Ester inRats

Chronic toxicity of S(+) Ibuprofen-L-Threonine ester was comparedagainst a vehicle, (+/−)racemic Ibuprofen and (+/−)racemicIbuprofen-L-Hydroproline ester. Test species used was Swiss Albino Mice,both male and female with body weight range of 18-27 gms. Randomizationwas done by the method of stratified randomization procedure using SASsoftware program (Version 8.2) with stratification by bodyweight. Thevarious groups are depicted below: TABLE 9 NUMBER OF ANIMAL NUMBERSGROUP TEST ITEM ANIMALS Female Male Vehicle Vehicle 10 01 to 05 06 to 10Control Test Group 1 L-Threonine 10 11 to 15 16 to 20 ester of S(+)Ibuprofen Test Group 2 L- 10 21 to 25 26 to 30 Hydroxyproline ester ofS(+) Ibuprofen Reference Test Ibuprofen USP 10 31 to 35 36 to 40 Group

The test doses are expressed as Ibuprofen molar equivalents TABLE 9ADose (mg per kg) Equivalent [in term of weight of the Test Item GroupIbuprofen] Test item [mg] Vehicle Vehicle 0.0 0.0 Control L-Threonineester of Test Group 1 200.0 334.0 S(+) Ibuprofen L-Hydroxyproline esterTest Group 2 200.0 310.0 of Racemic Ibuprofen Ibuprofen USP Reference200.0 200.0 (Racemic mixture) Control

The duration of dosing was 28 days. All the animals were daily till theend of the study for the presence/absence of clinical symptoms oftoxicity. Cage side observations included changes in the skin, eyes,posture, gait, respiration and behavior pattern. The incidence oftwitching, tremors, convulsions, salivation, diarrhea and death if any,were also recorded.

Animals exposed to different doses of the test substance did notindicate any symptoms of toxicity (Table 10). TABLE 10 Summary ofClinical Symptoms of Toxicity in Albino Mice Period of GROUP AnimalSigns in days (mg/kg body weight) Symptoms of toxicity Sex NumbersFrom-to Mortality Vehicle Control (0.0) No symptoms of Female 01 to 050-28 Nil (No Treatment) toxicity were observed No symptoms of Male  6 to10 0-28 Nil toxicity were observed Test Group 1 No symptoms of Female 11to 15 0-28 Nil (Ibuprofen S + T) toxicity were observed (334.0 mg/kg) Nosymptoms of Male 16 to 20 0-28 Nil toxicity were observed Test Group 2No symptoms of Female 21 to 25 0-28 1/5 (Ibuprofen HP) toxicity wereobserved (310.0 mg/kg) No symptoms of Male 26 to 30 0-28 Nil toxicitywere observed Reference Control 3 No symptoms of Female 31 to 35 0-283/5 (Ibuprofen USP) toxicity were observed (200.0 mg/kg) No symptoms ofMale 36 to 40 0-28 1/5 toxicity were observedIn the above table, Ibuprofen S + T refers to S(+)Ibuprofen-L-ThreonineEster and Ibupofen HP refers to Ibuprofen hydroxy Proline Ester.

Death Record

Ibuprofen HP

Animal no. 23 (female animal) died on 21 day of dosing.

Positive Control Group

Animal no. 31 (female animal) died on 23 day of dosing.

Animal no. 32 (female animal) died on 21 day of dosing.

Animal no. 33 (female animal) died on 24 day of dosing.

Animal no. 40 (male animal) died on 10 day of dosing.

While there were no cage side specific toxicity noted, surprisingly 40%of the animals receiving racemic ibuprofen died, only 10% of the ratsreceiving Hydroxyproline ester of Iburprofen did not complete the fullcourse, and even more surprisingly, none of the animals in theS(+)Ibuprofen-L-Threonine ester group died. The averageincrease/decrease is body weight, and percentage change of body weightof the surviving animals in various groups are shown below: TABLE 11Average Change Percentage Change Treatment In body Weight (gms) (No ofanimals survived) Vehicle 4.65 19.97 (10) S(+)Ibuprofen −0.68 −3.61 (10)Threonine Ester Ibuprofen Hydroxy- 1.43  5.75 (9) Proline Ester Racemic2.97 12.54 (6) Ibuprofen

While there was increase in body weight in treatments with racemicIbuprofen and Ibuprofen Hydroxyproline ester, both group havemortalities, with currently marketed Ibuprofen showing more mortalitythan Hydroxyproline ester. Hence all the amino acid esters are farsuperior to Ibuprofen racemic mixture or the active S(+)Ibuprofen.However, the best product so far seems to be S(+)Ibuprofen-L-ThreonineEster, making it one of the ideal candidates to be advanced to humantrials.

Human Clinical Trials with S(+)Ibuprofen-L-Threonine Ester:

Determination of the Analgesic and Anti-Inflammatory Effects ofS(+)Ibuprofen-L-Threonine Ester in Three Human Volunteers:

Two male, age 49 and 50 having severe headache took 1 capsule containingS(+)Ibuprofen-L-Threonine Ester. The capsule contents were equivalent to200 mg of racemic Ibuprofen. Relief from headache was reported after 15min, and complete absence of any pain from headache was reported at theend of 1 hour, which lasted for another 12 hours.

Two males age 49 and 51 took 1 capsule each containingS(+)Ibuprofen-L-Threonine ester for arthritic knee pain, which wasperceptible. After 12 hours, both volunteers reported significantreduction in the pain associated with their right knee. Suchamolearation of pain was further sustained for another 24 hours.

Pharmacokinetics of Ibuprofen in Human Volunteers:

Based upon preliminary analgesic and anti-inflammatory response from the4 volunteers, Ibuprofen racemic drug was compared againstS(+)Ibuprofen-L-Threonine ester at 200 mg equivalent dose. Theplasma-concentration time profile in 6 volunteers, whereS(+)Ibuprofen-L-Threonine ester concentrations were plotted againstracemic ibupronfe concentrations in plasma for each volunteers.

Based upon the results of comparative bioavailablity of racemicIbuprofen versus Ibuprofen released from S(+)Ibuprofen-L-Threonine esterit is clear that only very small amount of Ibuprofen is released intactinto human blood stream. This is due to the fact thatS(+)Ibuprofen-L-threonine ester does not act as a prodrug of Ibuprofen,instead the Threonine ester had intact activity.

FIG. 3-8 are plots of plasma concentration of Ibuprofen in humanvolunteers after administration of 200 mg (or equivalent) of racemicIbuprofen to human volunteers. The overall bioavailability of Ibuprofenfrom Ibuprofen Racemic mixture of 200 mg and equivalent dose ofS(+)Ibuprofen-L-Threonine ester are shown in the table below: TABLE 12Volunteer 111 AUC1 AUC2 % Availability 1 69197.618 893.226 1.3 241861.277 1978.925 4.7 3 73121.747 940.133 1.3 4 38993.502 2101.642 5.45 34567.246 1657.496 4.8 6 66710.152 925.000 1.4

In the above table, AUC1 represents the cumulative area under the plasmaconcentration time curve following oral administration of 200 mg ofracemic ibuprofen to human volunteers, and AUC2 represents thecumulative area under the plasma concentration time curve following oraladministration of 200 mg ibuprofen equivalence ofS(+)Ibuprofen-L-Threonine ester. The third column in the above tableshows relative bioavailability of Ibuprofen in human plasma after oraladministration of S(+)Ibuprofen-L-Threonine ester at equivalent doses.This clearly demonstrates that any activity seen in human volunteers isnot due to release of any significant amounts of S(+)Ibuprofen into theplasma after oral ingestion of the Threonine ester.

The reason that S(+)Ibuprofen advanced to human pharmacokinetic studieswas due to a lack of toxicity in 28-day chronic administration in ratscompared to Ibuprofen, or Hydroxyproline ester of Ibuprofen.Furthermore, earlier studies indicated that S(+)Ibuprofen is highlytoxic to gastric mucosa of rats. Similar results were also shown invarious studies elsewhere, for example, other investigators comparedS(+) and R(−) enantiomers of ibuprofen in male Wistar rats. At 40 mg/kgdose, microscopic evaluation of the GI tissue samples revealedsignificance difference in GI toxicity caused by S(+) than R(−)Ibuprofen (See Janjikhel, R K, Bricker, J D, Borochovitz, D, Adeyeye, CM, Stereoselective Disposition of Sustained Release Microspheres ofIbuprofen Enantiomers in Rats: II, Acute Gastrointestinal Toxicity. DrugDelivery, Vol 6, No. 3, August 1999, pp 163-170). HoweverS(+)Ibuprofen-L-Threonine ester was GI sparing, and had no toxicity.

Futhermore, it has been reported that R(−)Ibuprofen may be capable ofinhibiting both therapeutic and toxic effects of S(+)Ibuprofen (SeeKaehler, S T, Phleps, W, Hesse, E. Dexibuprofen: Pharmacology,therapeutic uses and safety. Infammopharmacology, Vol 11 No. 4-6, 2003,pp 371-383). This is also consistent with observation, since in acute GItoxicity studies racemic ibuprofen was somewhat less toxic thanS(+)Ibuprofen.

Similarly, Rainsford K D, in Pharmacology and Toxicology of Ibuprofen,in Rainsford, K D, ed. Ibuprofen, A critical bibliographic Review.London, Taylor and Francis, 2000, states that competition between theenantiomers of ibuprofen for prostaglandin production in vitro wasevident, and that inhibition of binding of S(+) ibuprofen by R(−)ibuprofen in the racemic mixture contributed to the GI tolerance of theracemate.

However, what is not known in the art is that an ester of S(+)Ibuprofenwould also be nontoxic. For example, one trained in the art of suchderivative pharmacology, would have concluded that as S(+)Ibuprofen ishighly toxic to the GI mucosa, and since S(+)Ibuprofen will be releasedfrom S(+)Ibuprofen-L-Threonine ester by the esterase enzymes in GItract, and by the action of pancreatin enzyme in the duodenum, onetrained in the art would have predicted that there will not be anyreduction in toxicity. However, the current inventor surprisingly notedthat S(+)Ibuprofen-L-Threonine ester is significantly and completelynon-toxic to GI mucosa in the rats tested.

In spite of no ibuprofen appearing in the human plasma after oraladministration of S(+)Ibuprofen-L-Threonine ester, significant analgesicand anti-inflammatory response was seen in the 2 volunteers each testedtwice. Thus, S(+)Ibuprofen-L-threonine ester does not act as a prodrug,and it seems to have intact pharmacological activity.

While less effectiveness was seen in rat model withS(+)Ibuprofen-L-Threonine ester, the overriding factor that demonstratethat this drug is more suitable for human treatment of various diseasessuch as arthritis etc., is due to the fact that on chronic toxicitytrials none of the animal died in this drug group. Furthermore, theS(+)Ibuprofen-L-Threonine ester exhibited no toxicity in either thegastric system or in the whole animal (as determined by number ofsurviving rats), thereby indicating that there is no toxicity potentialin systemic circulation of human subjects with respect to this threoninederivative. Thus, S(+)Ibuprofen-L-Threonine ester exhibits enhancedpharmacological action, such as observed analgesic, anti-inflammatoryand likely anti-pyretic properties elicited in humans.

Human Clinical Trial:

S(−)Ketorolac-L-Threonine ester capsules were filled using dextrose asthe filler. The Ketorolac-L-Threonine ester dose was comparable toracemic Ketorolac Tromethamine tablets. For example, 13 mg otS(−)Ketorolac-L-Threonine ester was roughly equivalent to 13 mg ofracemic Ketorolac tromethamine in tablet and/or capsule form.

A Female patient (age 72) suffereing from severe ankolysing spodolytis,arthritis and other inflammatory joint probems was under treatment withIndomethacin, 25 mg twice daily dose. In order the evaluate theanalgesic activity of S(−) Ketorolac-L-Threonine ester, the patient waswithdrawn from treatment of Indomethacin. After 24 hours, the pain wasreturning and she was administered with 13 mg of S(−) KetorolacL-Threonine ester, once in the morning and once in the evening. This wasrepeated for 5 days. During the entire period, the patient demonstratedlack of pain, no gastric irritation symptoms, or other side effects.

About 3 months later, the same above female volunteer repeated theexperiment. In this time, she went off the indomethacin, and wasadministered only one dose of 13 mg of S(−) Ketorolac-L-Threonine ester.After the 2^(nd) day, she complained of the pain resurfacing, and thedose was then increased on the morning of the 3^(rd) day to twice daily13 mg each. Beginning the 3^(rd) day she informed the doctor of lack ofany pain, and this treatment was continued for another 3 days at twocapsules of 13 mg each. On the 6^(th) day, morning she was then switchedback to Indomethacin. This study showed that in this particularvolunteer, 13 mg twice daily was the appropriate dose to alleviate hersevere pain.

There are a number of screening tests to determine the utility of thederivatives created according to the disclosed methods. These includeboth in vitro and in vivo screening methods.

Surprisingly better results with L-Threonine esters of various drugs,racemic or otherwise were obtained. For examples, as describedhereinabove, it was shown that the L-Threonine ester of Ibuprofen andKetorolac possessed good clinical activity, and were less toxic in theanimal models (Ibuprofen). Similar results were also obtained withrespect to studies done with L-Threonine esters of various other drugswhich were not racemic mixtures. Examples worth noting are Aspirin andFenofibric Acid.

Synthesis of the various amino acid esters of Aspirin and Fenofibricacid are described in U.S. Ser. No. 11/343,557 and WO 2005/046575, thecontents of both which are incorporated by reference in their entiretyherein.

Gastric Mucosa Irritation Potential of the L-serine, L-Threonine, andL-Hydroxyproline esters of Acetylsalicylic Acid Compared toAcetylsalicylic Acid:—

A study was conducted to determine the relative potential of varousderivatives of aspirin (L-serine, L-Threonine, and L-Hydroxyprolineesters of acetylsalicylic acid) to cause gastric mucosalirritation/lesions in fasted male albino rats. Aspirin served asreference control.

The amino acid esters of aspirin and aspirin were administered by gavageto fasted male albino rats (Wistar strain), using 0.5% (w/v)Carboxymethylcellulose (CMC) in Phosphate Buffer (pH 2.6) solution asthe vehicle. The study was conducted at two dose levels viz. 100 mg and200 mg/kg body weight along with a vehicle control group. At each doselevel 5 animals were used.

The rats were fasted for a period of 18 to 22 hours before dosing. Thetest item was administered as a single dose by gavage. Three hours afterdrug administration, the animals were killed humanely by CO₂ gasinhalation. The stomach was dissected out and observed for

-   -   the quantity of mucous exudate,    -   degree of hyperemia and thickening of stomach wall,    -   hemorrhagic spots (focal or diffuse), nature of hemorrhages        (petechial or ecchymotic) along with the size and    -   perforations

It was observed that none of the L-serine, L-Threonine, andL-Hydroxyproline esters of acetylsalicylic acid induced any evidence ofirritation of gastric mucosa at the two doses tested viz., 0.100 and 200mg/kg body weight. In contrast, aspirin (acetylsalicylic acid) causedirritation of the gastric mucosal in all the fasted male albino rats atthe dose level of 200 mg/kg.

However at the dose level of 100 mg/kg aspirin failed to cause anyevidence of gastric mucosal irritation in the male rats. Further none ofthe animals of different test groups showed any clinical symptoms oftoxicity throughout the observation period of three hours.

However, the Threonine ester is less toxic than the other esters of theother hydroxy containing amino acids, and thus has a more effectiverelative therapeutic index.

Preliminary Blood Clotting Time Efficacy Trials in Rodents:

Since Aspirin is associated with a number of toxicities, primarily GIirritation, bleeding, ulcer and hemorrhage, several amino acidderivatives of Aspirin were prepared.

Drug Derivatives Synthesized: a) Aspirin-L-Serine Ester; b)Aspirin-L-Threonine Ester; c) Aspirin-L-Hydroxyproline Ester.

Doses Used: 1, 4, 10 and 20 mg/kg (Aspirin Equivalence)

Observations of Blood Clotting Time:

The data on the mean clotting time (MCT) of the animals of low,intermediate and high dose groups of different formulations, vehiclecontrol and positive control groups estimated one hour after dosing werepresented below (Table 13 and FIGS. 9 to 14): TABLE 13 Summary of MeanClotting Time (± S.D.) in Minutes - L-serine, L-Threonine, andL-Hydroxyproline esters of acetylsalicylic acid and Aspirin (Positivecontrol) 1 mg/kg 4 mg/kg 10 mg/kg Vehicle control 4.9 ± 1.10 L-Serineester of 5.7 ± 1.34 6.8 ± 1.48 6.9 ± 1.37 acetylsalicylic acidL-Hydroxyproline ester 6.1 ± 1.10 5.7 ± 0.82 7.5 ± 1.18 ofacetylsalicylic acid L-Threonine, ester of 5.2 ± 1.14 5.6 ± 0.84 7.4 ±0.97 acetylsalicylic acid Positive control 6.2 ± 1.40 8.1 ± 1.97 9.8 ±1.32 (acetylsalicylic acid)The group mean data of animals comparing the Relative Efficacy ofL-serine, L-Threonine, and L-Hydroxyproline esters of acetylsalicylicacid to each and compared to acetylsalicylic acid on Mean Clotting Time(±S.D.) in Minutes is depicted in FIG. 9.

The statistical analysis of FIG. 9 showed that L-Threonine, andL-Hydroxyproline esters of acetylsalicylic acid are as effective asacetylsalicylic acid. There is no significant difference at 5%significance level for L-Hydroxyproline ester of acetylsalicylic acidand L-Threonine ester of acetylsalicylic with respect to positivecontrol for the mean blood clotting time observed after two hours.However, combined with the gastric irritation potential, the L-serine,L-Threonine, and L-Hydroxyproline esters of acetylsalicylic acid are farsuperior (See FIG. 10).

FIG. 10 shows a clotting time in minutes verses doses administered torats at 1, 4 and 10 mg/kg for test drugs ASA-Serine Ester,ASA-Hydroxyproline Ester and ASA-Threonine Ester versus Aspirin.Statistically significant increases clotting time was noted for all testand reference drugs at 10 mg/kg dose.

The following additional data were obtained, when the same drugs werecompared in rats on a different date with different time intervals anddoses: TABLE 14 Clotting Time (min) Dose ASA-Serine ASA-HP ASA-T ASAVehicle 10 mg/kg 1 hr 6.9 7.5 7.4 9.8 4.9  10 mg/kg 24 hr 4.4 3.3 3.64.4 2.7 20 mg/kg 2 hr 3.8 4.2 5.3 5.4 2.7

Two of the better esters of ASA at various conditions are as follows:

10 mg/kg 1 hr ASA-HP and ASA-T

10 mg/kg 24 hr ASA-S and ASA-T

20 mg/kg 2 hr ASA-HP and ASA-T

Thus Acetylsalicylic Acid-L-Threonine Ester (ASA-T) was the preferredderivative for treatment in the advanced Chronic 28 Day ToxicityStudies.

Note: The statistical analysis showed that L-Threonine, andL-Hydroxyproline esters of acetylsalicylic acid are as effective asacetylsalicylic acid. There is no significant difference at 5%significance level for L-Hydroxyproline ester of acetylsalicylic acidand L-Threonine ester of acetylsalicylic with respect to positivecontrol for the mean blood clotting time observed after two hours.However, combined with the gastric irritation potential, the L-serine,L-Threonine, and L-Hydroxyproline esters of acetylsalicylic acid are farsuperior. Furthermore, when compared against esters, L-Threonine Esterof Aspirin seems to be the ideal candidate as it showed consistentlybetter response in the rat blood clotting time.

Based upon consistently improved toxicity profile, efficacy and bettertherapeutic index, Acetylsalicylic Acid-L-Threonine Ester was advancedto GMP synthesis.

Results of the 28-Day Chronic Dosing in Rodents, Comparative Toxicology:

The purpose of this study is to establish the toxicity ofAcetylsalicylic Acid-L-Threonine Ester in relation to Aspirin (Make:Sigma, Batch number 090K0884) which served as a reference drug byconducting a 28-day repeated dose oral toxicity test in male and femalealbino rats.

Aspirin and Acetylsalicylic Acid-L-Threonine Ester were administered toalbino rats (Wistar strain), by oral gavage daily for a period of 28days, using 0.5% Carboxymethylcellulose (CMC) in phosphate buffersolution (pH 2.6) as vehicle. The study was conducted at one dose levelonly along with a vehicle control group as per the recommendation of theSponsor. The test doses are expressed as Aspirin molar equivalents.Acetylsalicylic Acid-L-Threonine Ester was compared against Aspirin andVehicle at 100 mg/kg dose administered to rats for 28 days.

The salient features of the study are as follows, where ASA-T representsAcetylsalicylic Acid-L-Threonine Ester:

-   -   1. All the animals of vehicle control group and the test group        (Acetylsalicylic Acid-L-Threonine Ester) and reference control        group (Aspirin) survived through the dosing period of 28 days.    -   2. None of the animals of the vehicle control group, test group        (Acetylsalicylic Acid-L-Threonine Ester), and reference control        group (Aspirin) exhibited any clinical symptoms of toxicity        through out the dosing period.

3. Changes in the Body weight. TABLE 15 Body Weight ComparisonSignificance[P < 0.05] Gain ASA-T vs Aspirin vs Vehicle Normal (Male)Gain ASA-T and Aspirin vs Vehicle Decrease (Female)The percentage decrease were 29% and 21% for AcetylsalicylicAcid-L-Threonine Ester and Aspirin

-   -   4. Food intake of the animals of both the sexes of test group        (Acetylsalicylic Acid-L-Threonine Ester) and reference control        group (Aspirin) was found to be normal and comparable to the        animals of vehicle control group.

5. Results of hematological analysis of the animals of different groupsare shown below: TABLE 16 Hematological Comparison Significance[P <0.05] All Blood Parameters ASA-T vs Aspirin vs None Vehicle PlateletCount ASA-T vs Aspirin Increase (Male)

6. Results of clinical chemistry analysis of the animals of differentgroups are summarized below: TABLE 17 Clinical Chemistry ComparisonSignificance[P < 0.05] Alkaline Phosphatase AST-T Ester vs VehicleIncrease (Female) Total Protein ASA-T Ester vs Vehicle Increase (Female)Creatinine ASA-T Ester vs Vehicle Decrease (Male) Cholesterol ASA-TEster vs Vehicle Increase (Male) Alkaline Phosphatase Aspirin vs VehicleIncrease (Female) Sodium Aspirin vs Vehicle Increase (Female) Blood UreaAspirin vs Vehicle Decrease (Female) SGPT and Cholesterol Aspirin vsVehicle Increase (Male) All Clinical Chemistry ASA-T Ester vs AspirinNone(but two below) Blood Glucose ASA-T Ester vs Aspirin Decrease(Female) Creatinine ASA-T Ester vs Aspirin Decrease (Male)

-   -   7. Necropsy of the surviving animals at the end of the study        (terminal necropsy) of vehicle control and different treatment        groups did not reveal any gross pathological changes in any of        the vital organs. Further there is no evidence of gastric        mucosal irritation in the animals of vehicle control, test group        (ASA-T Ester) and reference control group (Aspirin).

8. The data on absolute (Abs) and relative (Rel) organ weights of liver,kidney, adrenals, heart, spleen and testes showed the following changesin the organ weights: TABLE 18 Rel/Abs Organ Wts ComparisonSignificance[P < 0.05] Adrenals (Abs) ASA-T Ester vs Vehicle Decrease(Male) Kidney (Abs) Aspirin vs Vehicle Decrease (Male) Spleen (Abs)Aspirin vs Vehicle Decrease (Female) Kidney (Rel) Aspirin vs VehicleIncrease (Male) Spleen (Rel) Aspirin vs Vehicle Increase (Female) Kidney(Abs) ASA-T Ester vs Aspirin Decrease (Male) Spleen (Abs) ASA-T Ester vsAspirin Decrease (Male) Kidney (Rel) ASA-T Ester vs Aspirin Increase(Male)

-   -   9. Histological sections of the following organs viz. brain,        stomach, small intestines, large intestine, liver, kidney,        adrenal, spleen, heart, lungs and gonads of male and female        animals treated with Acetylsalicylic Acid-L-Threonine Ester or        reference drug (Aspirin) groups did not show any        histopathological changes and were found to be normal and        comparable to that of animals of vehicle control group. However        few animals treated with reference drug (Aspirin) showed mild        fatty changes in the cardiac muscle fibers of heart and mild        catarrhal changes of gastric mucosa.

Human Clinical Trials:

Several blood clotting time studies were done in a limited number ofhuman volunteers with Acetylsalicylic Acid-L-Threonine Ester. Resultsare shown in FIGS. 11-14.

Acetylsalicylic Acid-L-Threonine Ester at low dose of 80 mg was aseffective as 325 mg Bayer Aspirin (with baseline correction)! And, it ismore effective than 81 mg Bayer Aspirin as the results in FIG. 11 show.

FIG. 11 depicts Average Clotting Time after 325 mg and 81 mgAcetysalicylic Acid-L-Threonine Ester and Bayer Aspirin is administeredto human Volunteers. The first set of columns (Normal) is averageclotting times observed prior to administration of the Aspirinderivative and Aspirin—This data is given in table 19 below.

Table 19 shows the Comparative average clotting time (min) details ofAcetylsalicylic Acid-L-Threonine Ester vs. Bayer ASA at 325 mg and 81 mglevels. TABLE 19 AVERAGE CLOTTING TIME (MIN) 325 mg 81 mg 81 mg ASA-T325 mg ASA-T 81 mg ASA-T ESTER ASA Bayer ESTER ASA Bayer ESTER Volunteer1 Volunteer 2 Volunteer 1 Volunteer 2 Volunteer 3 Normal 4.5 4.5 4   4  4   With ASA-T ESTER 6.2 6.0 5.2 NA 5.1 With ASA (Bayer) 5.7 5.7 NA 4.5NA

FIG. 12 shows the clotting time in minutes—5 day administration of 81 mgAcetylsalicyclic Acid L-Threonine Ester and Bayer Aspirin in humanvolunteers. A total of three volunteers participated in the study, twotook Acetylsalicyclic Acid L-Threonine ester (bottom line in FIG. 12).

FIG. 13 graphically depicts the percentage increase in clotting time ofthe Acetylsalicyclic Acid L-Threoninr Ester relative to Aspirin (Bayer)at 81 mg dose based on a five day average increase. This plot wasderived from Table 13. The two acetylsalicyclic L-Threonine estersblocks shown in FIG. 13 correspond to two separate volunteers who tookthe test drug over a period of five days. The third volunteer took BayerAspirin for five days. As shown hereinbelow, an increase in clottingtime occurred on the very first day of drug intake and remained higherthan Bayer ASA during the subsequent administrations

FIG. 14 shows pharmacokinetic results in 4 volunteers who tookAcetylsalicylic Acid-L-Threonine Ester versus two volunteers who tookBayer Aspirin at 325 mg dose.

Therefore, as shown by the data, Acetylsalicylic Acid-L-Threonine Esteris a Superior Anti platelet drug:

-   -   1. Does not produce any gastric irritation;    -   2. Does not inhibit prostaglandin synthesis;    -   3. Does not have any COX-1 or COX-2 activity on the endothelial        or vascular tissues, as no ASA from Acetylsalicylic        Acid-L-Threonine Ester reaches systemic circulation.    -   4. Thus, none of the side effects of Aspirin are seen with        Acetylsalicylic Acid-L-Threonine Ester.

FIG. 15 graphically depicts a plot of plasma concentration of salicyclicAcid versus time in human plasma in four volunteers who ingestedAcetylsalicyclic Acid-L-Threonine ester and two volunteers who ingestedaspirin.

Rapid influx and efflux of Salicylic Acid from Aspirin administrationwas seen in the two volunteers who took Aspirin (FIG. 15). There wasprolonged and sustained formation and disappearance of Salicylic acid involunteers who took Acetylsalicylic Acid-L-Threonine Ester (FIG. 15).This is indicative of the fact the there is a high likelihood ofAcetylsalicylic Acid-L-Threonine Ester exhibiting sustained, specificand irreversible acetylation of the platelets in the portal circulation.Since there was no aspirin found in the systemic circulation after oraldosing of Acetylsalicylic Acid-L-Threonine Ester, it is likely that sitespecific action of acetylation of the platelets has been achieved, andthe unwanted effect of aspirin in the endothelial system has beenavoided. Thus it is evident from the human pharmacokinetic studies,clinical trials, rat gastric mucosa irritation results, AcetylsalicylicAcid-L-Threonine Ester is a superior anti-platelet drug that does nothave the toxicity of Aspirin and also has a better Therapeutic Index.

Anti-Hyperlipidemic Effect of Fenofibric Acid Derivatives:

Surprisingly, the L-threonine derivatives of Fenofibric acids made areeither equipotent or showed better efficacy in rat anti-hyperlipidemicmodel. In this study, Albino rats were placed on a hypertriglyceridemicdiet, viz., 30% sucrose in drinking water for a period 7 days. Thesucrose solution was supplied ad libitum along with normal diet. Albinorats were administered with appropriate daily dose of the test itemdaily for a period of 7 days from day 8 to 14. During this period ratsare provided with normal diet only. Blood samples were collected on dayzero (before providing sucrose solution) on day 7 (before the start offirst dose) and on day 14 (end of the study). Serum was separated andanalyzed for triglycerides. Basing on the data the hypolipidemicproperty of the ester of Fenofibric acid were evaluated with referenceto fenofibrate (reference control). The results are shown in thefollowing table, where Fen-S Ester, Fen-T Ester and Fen-HP Ester areFenofibric Acid L-Serine Ester, Fenofibric Acid L-Threonine ester andFenofabric Acid L-Hydroxyproline ester respectively. TABLE 20Anti-lipidemic Effects of Fenofibric Acid and its Derivatives Absolute %% Absolute Change Change Change Dose Change from from from mg/kg fromday 0 day 7 day 0 day 7 Vehicle 50.4 −5.6 83.17% −4.80% Fen-S 25 −54 −90−61.64% −72.82% Ester 50 −31.4 −84.2 −44.10% −67.90% 100 −20.8 −72−33.23% −63.27% Fen-T 25 −18.2 −36.4 −23.21% −37.68% Ester 50 −23.8−77.6 −35.00% −63.71% 100 −63.8 −88.4 −68.45% −75.04% Fen-HP 25 −16−47.8 −32.92% −59.45% Ester 50 −35.8 −70.8 −49.31% −65.80% 100 −3.4 −112−7.52% −72.82% Fenofibrate 25 −10.8 −51 −15.21% −45.86% 50 −13.4 −87.6−22.95% −66.06% 100 −40.8 −71.6 −61.26% −73.51%

While all of the esters were active and showed efficacy, there wereimportant distinguishing factors between the various esters andFenofibrate. For example, dose dependent decrease in triglycerides werenoted with L-Threonine ester, and also maximum decrease from baselinelevel and treatment level were also noted for this compound. ThusFenofibric Acid-L-Threonine ester had overall superioranti-hyperlipidemic properties.

The in vitro methods include acid/base hydrolysis of the derivatives,hydrolysis in pig pancreas hydrolysis in rat intestinal fluid,hydrolysis in human gastric fluid, hydrolysis, as described in Simmons,D M, Chandran, V R and Portmann, G A, Danazol L-Threonine Derivatives:In Vitro and In Situ Biopharmaceutical Evaluation, Drug Development andIndustrial Pharmacy, Vol 21, Issue 6, Page 687, 1995, the contents ofall of which are incorporated by reference.

The compounds of the present invention are effective in treatingdiseases or conditions in which NSAIDs normally are used. Thederivatives disclosed herein are transformed within the body to releasethe active compound and enhances the therapeutic benefits of the NSAIDsby reducing or eliminating biopharmaceutical and pharmacokeneticbarriers associated with each of them. However it should be noted thatthese derivatives themselves will have sufficient activity withoutreleasing any active drug in the mammals. Since the derivatives is moresoluble in water then Ibuprofen or other NSAIDs, it does not need to beassociated with a carrier vehicle, such as alcohol or castor oil whichmay be toxic or produce unwanted side reactions. Moreover, oralformulations containing the NSAID derivatives are absorbed into theblood and are quite effective.

Thus, the derivatives of the present invention enhance the therapeuticbenefits by removing biopharmaceutical and pharmacokenetic barriers ofexisting drugs.

Furthermore, these derivatives are easily synthesized in high yieldsusing reagents which are readily and commercially available.

As defined herein, the term lower alkyl refers to an alkyl groupcontaining 1-6 carbon atoms. The alkyl groups may be straight chained orbranched. Examples include, methyl, ethyl, propyl, isopropyl, n-butyl,sec butyl, isobutyl, t-butyl, n-pentyl amyl, isopentyl, hexyl and thelike. The preferred lower alkyl group is methyl.

The term aryl refers to an aromatic ring containing only carbon ringatoms and having 6, 10 or 14 ring carbon atoms and up to a total of 18carbon atoms. It may be moncyclic or bicyclic or tricyclic. If itcontains more than 1 ring, the rings are all fused, but all of the ringsare fully aromatic. Examples include phenyl, α-naphthyl, B-naphthyl andthe like. The preferred aryl group is aryl.

Examples of aryl lower alkyl include benzyl, phenethyl, naphthylmethyl,naphthylethyl and the like.

The term cycloalkyl, as used herein, refers to an alicyclic hydrocarboncontaining 3-14 ring carbon atoms and up to a total of 18 carbon atoms.It may be monocyclic or it may be bicyclic, tricyclic or tetracyclic. Ifit contains more than 1 ring, the rings are fused to each other. Thecycloalkyl group may be fully saturated or partially saturated. It alsomay contain an aromatic moiety ring. Examples include cycloproyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl,norbornyl, adamantyl, indanyl, and the like. The preferred cycloalkylcontains 5-8 ring carbon atoms and especially 5-6 ring carbon atoms.

Examples of cycloalkyl lower alkyl include cyclohexylmethyl,cyclohexylethyl, trans-1,3-dimethylcyclohxyl, trans1,3-dimethyl-cyclpentyl and the like.

The term heterocyclic refers to a cycloalkyl or aryl ring, as definedherein, wherein at least one of the ring carbon atoms has beensubstituted with a heteratom selected from nitrogen, sulfur or oxygen.The term heterocycles also includes the heteroaramatics. The heterocyclegroup may contain 1, 2, 3 or 4 ring heteratoms, but preferably 1 or 2ring heteroatoms. Examples include furyl, tetrahydrofuryl, pyrridyl,pyrryl, thienyl, pyrazolyl, pyrrolyl, imidazolyl, indolyl, thiazolyl,oxazolyl, isothiazolyl, isoxazolyl, piperidyl, pyrrolinyl, piperazinyl,quinolyl, triazolyl, tetrazolyl, isoquinolyl, benzofuryl, benzothienyl,morpholinyl, benzoxazolyl, tetrahydrofuryl, purinyl, indolinyl,pyrazolindinyl, imidazolinyl, imadazolindinyl, pyrrolidinyl, furazanyl,N-methylindolyl, methylfuryl, pyridazinyl, pyrimidinyl, pyrazinyl,pyridyl, aziridino, oxetanyl, azetidinyl, the N-oxides of the nitrogencontaining heterocycles, such as the nitric oxides of pyridyl,pyrazinyl, and pyrimidinyl and the like.

The terms “compounds of the present invention”, “derivatives of thepresent invention”, “drugs of the present invention” are usedinterchangeably. They refer to the drugs in which a L-Threonine moietyas described herein, is covalently bound to a drug or medicament.

The term ASA refers to acetyl salicyclic acid.

As used herein, the term “cage size toxicity” has been used with respectto the administration of ibuprofen and/or its derivatives. What thisterm refers to is that a number of animals died during the 28-daytoxicity studies. However, in most instances there were no visible signsof impending death, such as lethargy, significant weight loss, loss ofmobility, and other outward signs of approaching death that were noted.Therefore it was not easy to predict which of the animals would die.When the cages were opened, the next morning one or more animals werenoted to be dead. Thus visual observation of toxicity of animals on thecage is also noted as “cage side specific toxicity”.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for enhancing at least one or more of the therapeuticqualities of a drug having a functionality group selected form the groupconsisting of hydroxy, amino, carboxy or acylating derivatives of saidcarboxy group, said improved therapeutic quality being selected from thegroup consisting of: (a) improved taste or smell (b) desiredoctanol/water partition coefficient (c) improved stability (d) enhancedpenetration of blood-brain barrier (e) elimination of first pass effectin the liver (f) reduction of intero-hepatic recirculation (g) painlessinjection with parental formulation (h) improved bioavailability (i)improved changes in the rate of absorption (j) reduced side effects (k)dose proportionality (l) selective hydrolysis of the derivative at siteof action (m) controlled release properties (n) targeted drug delivery(o) reduction in toxicity (p) reduced dose (q) alteration of metabolicpathway to deliver more drug at site of action (r) increased solutilityin aqueous solution and (s) enhanced efficacy, the method comprising (a)reacting the drug with naturally occuring L-Threonine amino acid underconditions effective to form a covalent bond between the drug and theL-Threonine and (b) administering the product of (a) to a patient inneed thereof.
 2. The method according to claim 1 wherein the drug isAbacavir, Acarbose, Acebutolol, Acetaminophen, Adefovir, Albuterol,Amlodipinel, Amoxicillin, Amphotericin B, Amprenavir, Aspirin, Atenolol,Atorvastatin, Atropine, Atovaquone, Baclofen, Benazeprilat, Betaxolol,Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol, Bitolterol,Brinzolamide, Bupivacaine, Buprenorphine, Bupropion, Butorphanol,Candesartan, Capacitabine, Captopril, Carbidopa, Carnitine, Carteolol,Carvedilol, Cefdinir, Cefditoren, Ceftazimide, Cefpodoxime, Cefuroxime,Cerivastatin, Chloramphenicol, Ciprofloxacin, Cisapride, ClopidogrelAcid, Clorazepic Acid, Cycloserine, Cyclosporine, Cytarabine, Danazol,Dextroamphetamine, Diclofenac, Didanosine, Digoxin, Divalproex,Docetaxel, Dorzolamide, Dyphylline, Dysopyramide, Efavirenz,Enalaprilat, Ephedrine, Eplerenone, Eprosartan, Esmolol, Estramustine,Ethambutol, Ethchlorvynol, Ethosuximide, Ethotoin, Etidocaine,Etoposide, Ezetimibe, Famciclovir, Fenofibrate, Fenoprofen,Fexofenadine, Finasteride, Flavoxate, Fluoxetine, Flurbiprofen,Fluticasone, Fluvastatin, Folic Acid, Fosinoprilat, Frovatriptan,Fulvestrant, Gabapentin, Ganciclovir, Glimepiride, Goserelin,Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,Indapamide, Indinavir, Ipratropium, Iinotecan, Isosorbide, Isradipine,Ketoprofen, Ketorolac, Labetalol, Lamivudine, Lamivudine, Lansoprazole,Latanoprost Acid, Leuprolide, Levobunolol, Levodopa, Levorphanol,Liothyronine, Lisinopril, Lopinavir, Lorazepam, Lovastatin,Medroxyprogesterone, Mefloquine, Megestrol, Mephobarbital, Mepivacaine,Metaproterenol, Metformin, Methamphetamine, Methohexital, Methotrexate,Methylphenidate, Methylphenidate, Methylprednisolone, Metolazone,Metoprolol, Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,Nadolol, Nalbuphine, Naproxen, Naratriptan, Nateglinide, Nelfinavir,Niacin, Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,Ofloxacin, Olmesartan, Omeprazole, Paclitaxel, Pantothenic Acid,Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,Penciclovir, Pentazocine, Pentobarbital, Perindoprilat, Phenylephrine,Phenylpropanolamine, Pindolol, Pioglitazone, Pirbuterol, Pramipexole,Pravastatin, Propafenone, Propofol, Propoxyphene, Propranolol,Pseudoephedrine, Quinacrine, Quinaprilat, Quinethazone, Quinidine,Quinine, Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,Ritonavir, Ropivacaine, Rosiglitazone, Rosuvastatin, Salmeterol,Salsalate, Sertraline, Simavastatin, Sirolimus, Sotalol, Sulfa Drugs,Sulfasalazine, Sumitriptan, Tacrolimus, Tazorotene, Telmesartan,Tenofovir, Terbutaline, Thyroxine, Tiagabine, Timolol, Tirofiban,Tocainide, Tramadol, Trandolaprilat, Tranylcypromine, Treprostinil,Triamcinolone, Troglitazone, Unoprostone, Valsartan, Venlafaxine,Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton andZolmitriptan.
 3. The method according to claim 1 wherein the drug iscovalently bound to the Threonine via an ester or amide linkage.
 4. Theproduct from the reaction of a drug containing a functional groupselected from the group consisting of carboxyl or an acylatingderivative thereof, an amino group and a hydroxy group thereon and aL-Threonine amino acid.
 5. L-Threonine ester of a drug wherein the drugis Abacavir, Acarbose, Acebutolol, Acetaminophen, Adefovir, Albuterol,Amlodipinel, Amoxicillin, Amphotericin B, Amprenavir, Aspirin, Atenolol,Atorvastatin, Atropine, Atovaquone, Baclofen, Benazeprilat, Betaxolol,Bexarotene, Bicalutamide, Biotin, Biperiden, Bisoprolol, Bitolterol,Brinzolamide, Bupivacaine, Buprenorphine, Bupropion, Butorphanol,Candesartan, Capacitabine, Captopril, Carbidopa, Carnitine, Carteolol,Carvedilol, Cefdinir, Cefditoren, Ceftazimide, Cefpodoxime, Cefuroxime,Cerivastatin, Chloramphenicol, Ciprofloxacin, Cisapride, ClopidogrelAcid, Clorazepic Acid, Cycloserine, Cyclosporine, Cytarabine, Danazol,Dextroamphetamine, Diclofenac, Didanosine, Digoxin, Divalproex,Docetaxel, Dorzolamide, Dyphylline, Dysopyramide, Efavirenz,Enalaprilat, Ephedrine, Eplerenone, Eprosartan, Esmolol, Estramustine,Ethambutol, Ethchlorvynol, Ethosuximide, Ethotoin, Etidocaine,Etoposide, Ezetimibe, Famciclovir, Fenofibrate, Fenoprofen,Fexofenadine, Finasteride, Flavoxate, Fluoxetine, Flurbiprofen,Fluticasone, Fluvastatin, Folic Acid, Fosinoprilat, Frovatriptan,Fulvestrant, Gabapentin, Ganciclovir, Glimepiride, Goserelin,Hydroxychloroquine, Hydroxyzine, Hyoscyamine, Ibuprofen, Ibutilide,Indapamide, Indinavir, Ipratropium, Irinotecan, Isosorbide, Isradipine,Ketoprofen, Ketorolac, Labetalol, Lamivudine, Lamivudine, Lansoprazole,Latanoprost Acid, Leuprolide, Levobunolol, Levodopa, Levorphanol,Liothyronine, Lisinopril, Lopinavir, Lorazepam, Lovastatin,Medroxyprogesterone, Mefloquine, Megestrol, Mephobarbital, Mepivacaine,Metaproterenol, Metformin, Methamphetamine, Methohexital, Methotrexate,Methylphenidate, Methylphenidate, Methylprednisolone, Metolazone,Metoprolol, Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,Nadolol, Nalbuphine, Naproxen, Naratriptan, Nateglinide, Nelfinavir,Niacin, Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,Ofloxacin, Olmesartan, Omeprazole, Paclitaxel, Pantothenic Acid,Paroxetine, Paroxetine, Pemoline, Penbutolol, Penicillamine,Penciclovir, Pentazocine, Pentobarbital, Perindoprilat, Phenylephrine,Phenylpropanolamine, Pindolol, Pioglitazone, Pirbuterol, Pramipexole,Pravastatin, Propafenone, Propofol, Propoxyphene, Propranolol,Pseudoephedrine, Quinacrine, Quinaprilat, Quinethazone, Quinidine,Quinine, Ramiprilat, Reboxetine, Repaglinide, Repaglinide, Ribavirin,Ritonavir, Ropivacaine, Rosiglitazone, Rosuvastatin, Salmeterol,Salsalate, Sertraline, Simavastatin, Sirolimus, Sotalol, Sulfa Drugs,Sulfasalazine, Sumitriptan, Tacrolimus, Tazorotene, Telmesartan,Tenofovir, Terbutaline, Thyroxine, Tiagabine, Timolol, Tirofiban,Tocainide, Tramadol, Trandolaprilat, Tranylcypromine, Treprostinil,Triamcinolone, Troglitazone, Unoprostone, Valsartan, Venlafaxine,Vidarabine, Warfarin, Zalcitabine, Zidovudine, Zileuton andZolmitriptan.
 6. A pharmaceutical composition comprising the productproduced in claim 4 and a pharmaceutical carrier therefor.
 7. Apharmaceutical composition comprising the L-Threonine ester of the drugof claim 5 and a pharmacetical carrier therefor.
 8. A method offacilitating the seperation of a racemic mixture of a drug containing acarboxy, amino or hydroxy group into its separate stereoisomers whichcomprises reacting said drug with L-Threonine or acylating derivativethereof under conditions effective to form a covalent bond between thedrug and the L-Threonine or acylating derivative, and separating thestereoisomers thus formed.